Summary: DNA double-strand break repair and V(D)J recombination protein XRCC4
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XRCC4 Edit Wikipedia article
|X-ray repair complementing defective repair in Chinese hamster cells 4|
PDB rendering based on 1fu1.
|RNA expression pattern|
DNA repair protein XRCC4 also known as X-ray repair cross-complementing protein 4 or XRCC4 is a protein that in humans is encoded by the XRCC4 gene. In addition to humans, the XRCC4 protein is also expressed in many other metazoans, fungi and in plants. The X-ray repair cross-complementing protein 4 is one of several core proteins involved in the non-homologous end joining (NHEJ) pathway to repair DNA double strand breaks (DSBs).
NHEJ requires two main components to achieve successful completion. The first component is the cooperative binding and phosphorylation of artemis by the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). Artemis cleaves the ends of damaged DNA to prepare it for ligation. The second component involves the bridging of DNA to DNA Ligase IV (LigIV), by XRCC4, with the aid of Cernunos-XLF. DNA-PKcs and XRCC4 are anchored to Ku70 / Ku80 heterodimer, which are bound to the DNA ends.
Since XRCC4 is the key protein that enables interaction of LigIV to damaged DNA and therefore ligation of the ends, mutations in the XRCC4 gene were found to cause embryonic lethality in mice and developmental inhibition and immunodeficiency in humans. Furthermore certain mutations in the XRCC4 gene are associated with an increased risk of cancer.
- 1 Double strand breaks
- 2 Properties
- 3 Mechanism
- 4 Pathology
- 5 Anti-XRCC4 antibodies
- 6 History
- 7 References
- 8 Further reading
- 9 External links
Double strand breaks
DSBs are mainly caused by free radicals generated from ionizing radiation in the environment and from by-products released continually during cellular metabolism. DSBs that are not efficiently repaired may result in the loss of important protein coding genes and regulatory sequences required for gene expression necessary for the life of a cell. DSBs that cannot rely on a newly copied sister chromosome generated by DNA replication to fill in the gap will go into the NHEJ pathway. This method of repair is essential as it is a last resort to prevent loss of long stretches of the chromosome. NHEJ is also used to repair DSBs generated during V(D)J recombination when gene regions are rearranged to create the unique antigen binding sites of antibodies and T-cell receptors.
Sources of DNA damage
DNA damage occurs very frequently and is generated from exposure to a variety of both exogenous and endogenous genotoxic sources. One of these include ionizing radiation, such as Î³ radiation and X-rays, which ionize the deoxyribose groups in the DNA backbone and can induce DSBs. Reactive oxygen species, ROS, such as superoxide (O2â â¢ ), hydrogen peroxide (H2O2), hydroxyl radicals (HOâ¢), and singlet oxygen (1O2), can also produce DSBs as a result of ionizing radiation as well as cellular metabolic processes that are naturally occurring. DSBs can also be caused by the action of DNA polymerase while attempting to replicate DNA over a nick that was introduced as a result of DNA damage.
Consequences of DSBs
There are many types of DNA damage, but DSBs, in particular, are the most harmful as both strands are completely disjointed from the rest of the chromosome. If an efficient repair mechanism does not exist, the ends of the DNA can eventually degrade, leading to a permanent loss of sequence. A double-stranded gap in DNA will also prevent replication from proceeding, resulting in an incomplete copy of that specific chromosome, targeting the cell for apoptosis. As with all DNA damage, DSBs can introduce new mutations that can ultimately lead to cancer.
DSB repair methods
There are two methods for repairing DSBs depending on when the damage occurs during mitosis. If the DSB occurs after DNA replication has completed proceeding S phase of the cell cycle, the DSB repair pathway will use homologous recombination by pairing with the newly synthesized daughter strand to repair the break. However, if the DSB is generated prior to synthesis of the sister chromosome, then the template sequence that is required will be absent. For this circumstance, the NHEJ pathway provides a solution for repairing the break and is the main system used to repair DSBs in humans and multicellular eukaryotes. During NHEJ, very short stretches of complementary DNA, 1 bp or more at a time, are hybridized together, and the overhangs are removed. As a result, this specific region of the genome is permanently lost and the deletion can lead to cancer and premature aging.
Gene and protein
The human XRCC4 gene is located on chromosome 5, specifically at 5q14.2. This gene contains eight exons and three mRNA transcript variants, which encode two different protein isoforms. Transcript variant 1, mRNA, RefSeq NM_003401.3, is 1688 bp long and is the shortest out of the three variants. It is missing a short sequence in the 3â coding region as compared to variant 2. Isoform 1 contains 334 amino acids. Transcript variant 2, mRNA, RefSeq NM_022406, is 1694 bp long and encodes the longest isoform 2, which contains 336 amino acids. Transcript variant 3, RefSeq NM_022550.2, is 1735 bp and is the longest, but it also encodes for the same isoform 1 as variant 1. It contains an additional sequence in the 5âUTR of the mRNA transcript and lacks a short sequence in the 3â coding region as compared to variant 2.
XRCC4 protein is a tetramer that resembles the shape of a dumbbell containing two globular ends separated by a long, thin stalk. The tetramer is composed of two dimers, and each dimer is made up of two similar subunits. The first subunit (L) contains amino acid residues 1 â 203 and has a longer stalk than the second subunit (S) which contains residues 1 â 178.
The globular N-terminal domains of each subunit are identical. They are made up of two, antiparallel beta sheets that face each other in a beta sandwich-like structure (i.e., a "flattened" beta barrel) and are separated by two alpha helices on one side. The N-terminus begins with one beta sheet composed of strands 1, 2, 3, and 4, followed by a helix-turn-helix motif of the two alpha helices, Î±A and Î±B, which continues into strands 5, 6, 7, and ending with one alpha-helical stalk at the C-terminus. Î±A and Î±B are perpendicular to one another, and because one end of Î±B is partially inserted between the two beta sheets, it causes them to flare out away from each other. The beta sandwich structure is held together through three hydrogen bonds between antiparallel strands 4 and 7 and one hydrogen bond between strands 1 and 5.
The two helical stalks between subunits L and S intertwine with a single left-handed crossover into a coiled-coil at the top, near the globular domains forming a palm tree configuration. This region interacts with the two alpha helices of the second dimer in an opposite orientation to form a four-helix bundle and the dumbbell-shaped tetramer.
In order for XRCC4 to be sequestered from the cytoplasm to the nucleus to repair a DSB during NHEJ or to complete V(D)J recombination, post-translational modification at lysine 210 with a small ubiquitin-related modifier (SUMO), or sumoylation, is required. SUMO modification of diverse types of DNA repair proteins can be found in topoisomerases, base excision glycosylase TDG, Ku70/80, and BLM helicase. A common conserved motif is typically found to be a target of SUMO modification, Î¨KXE (where Î¨ is a bulky, hydrophobic amino acid). In the case of the XRCC4 protein, the consensus sequence surrounding lysine 210 is IKQE. Chinese hamster ovary cells, CHO, that express the mutated form of XRCC4 at K210 cannot be modified with SUMO, fail recruitment to the nucleus and instead accumulate in the cytoplasm. Furthermore, these cells are radiation sensitive and do not successfully complete V(D)J recombination.
Upon generation of a DSB, Ku proteins will move through the cytoplasm until they find the site of the break and bind to it. Ku recruits XRCC4 and Cer-XLF and both of these proteins interact cooperatively with one another through specific residues to form a nucleoprotein pore complex that wraps around DNA. Cer-XLF is a homodimer that is very similar to XRCC4 in the structure and size of its N-terminal and C-terminal domains. Residues arginine 64, leucine 65, and leucine 115 in Cer-XLF interact with lysines 65 and 99 in XRCC4 within their N-terminal domains. Together they form a filament bundle that wraps around DNA in an alternating pattern. Hyper-phosphorylation of the C-terminal alpha helical domains of XRCC4 by DNA-PKcs facilitates this interaction. XRCC4 dimer binds to a second dimer on an adjacent DNA strand to create a tetramer for DNA bridging early on in NHEJ. Prior to ligation, Lig IV binds to the C-terminal stalk of XRCC4 at the site of the break and displaces the second XRCC4 dimer. The BRCT2 domain of Lig IV hydrogen bonds with XRCC4 at this domain through multiple residues and introduces a kink in the two alpha helical tails. The helix-loop-helix clamp connected to the BRCT-linker also makes extensive contacts.
The process of NHEJ involves XRCC4 and a number of tightly coupled proteins acting in concert to repair the DSB. The system begins with the binding of one heterodimeric protein called Ku70/80 to each end of the DSB to maintain them close together in preparation for ligation and prevent their degradation. Ku70/80 then sequesters one DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the DNA ends to enable the binding of Artemis protein to one end of each DNA-PKcs. One end of the DNA-PKcs joins to stabilize the proximity of the DSB and allow very short regions of DNA complementarity to hybridize. DNA-PKcs then phosphorylates Artemis at a serine/threonine to activate its exonuclease activity and cleave nucleotides at the single strand tails that are not hybridized in a 5â to 3â direction. Two XRCC4 proteins are post-translationally modified for recognition and localization to Ku70/80 (5). The two XRCC4 proteins dimerize together and bind to Ku70/80 at the ends of the DNA strands to promote ligation. XRCC4 then forms a strong complex with DNA ligase IV, LigIV, which is enhanced by Cernunnos XRCC4-like factor, Cer-XLF. Cer-XLF only binds to XRCC4 without direct interaction with LigIV. LigIV then joins the DNA ends by catalyzing a covalent phosphodiester bond.
V(D)J recombination is the rearrangement of multiple, distinct gene segments in germ-line DNA to produce the unique protein domains of immune cells, B cells and T cells, that will specifically recognize foreign antigens such as viruses, bacteria, and pathogenic eukaryotes. B cells produce antibodies that are secreted into the bloodstream and T cells produce receptors that once translated are transported to the outer lipid bilayer of the cell. Antibodies are composed of two light and two heavy chains. The antigen binding site consists of two variable regions, VL and VH. The remainder of the antibody structure is made up of constant regions, CL, CH, CH2 and CH3. The Kappa locus in the mouse encodes an antibody light chain and contains approximately 300 gene segments for the variable region, V, four J segments than encode a short protein region, and one constant, C, segment. To produce a light chain with one unique type of VL, when B cells are differentiating, DNA is rearranged to incorporate a unique combination of the V and J segments. RNA splicing joins the recombined region with the C segment. The heavy chain gene also contain numerous diversity segments, D, and multiple constant segments, CÎ¼, CÎ´, CÎ³, CÎµ, CÎ±. Recombination occurs in a specific region of the gene that is located between two conserved sequence motifs called recombination signal sequences. Each motif is flanked by a 7 bp and 9 bp sequence that is separated by a 12 bp spacer, referred to as class 1, or a 23 bp spacer, referred to as class 2. A recombinase made up of RAG1 and RAG2 subunits always cleave between these two sites. The cleavage results in two hairpin structures for the V and J segments, respectively, and the non-coding region, are now separated from the V and J segments by a DSB. The hairpin coding region goes through the process of NHEJ where the closed end is cleaved and repaired. The non-coding region is circularized and degraded. Thus, NHEJ is also important in the development of the immune system via its role in V(D)J recombination.
Recent studies have shown an association between XRCC4 and potential susceptibility to a variety of pathologies. The most frequently observed linkage is between XRCC4 mutations and susceptibility to cancers such as bladder cancer, breast cancer, and lymphomas. Studies have also pointed to a potential linkage between XRCC4 mutation and endometriosis. Autoimmunity is also being studied in this regard. Linkage between XRCC4 mutations and certain pathologies may provide a basis for diagnostic biomarkers and, eventually, potential development of new therapeutics.
XRCC4 polymorphisms have been linked to a risk of susceptibility for cancers such as bladder cancer, breast cancer, prostate cancer, hepatocellular carcinoma, lymphomas, and multiple myeloma. With respect to bladder cancer, for example, the link between XRCC4 and risk of cancer susceptibility was based on hospital-based case-control histological studies of gene variants of both XRCC4 and XRCC3 and their possible association with risk for urothelial bladder cancer. The linkage with risk for urothelial bladder cancer susceptibility was shown for XRCC4, but not for XRCC3 With regard to breast cancer, the linkage with "increased risk of breast cancer" was based on an examination of functional polymorphisms of the XRCC4 gene carried out in connection with a meta-analysis of five case-control studies . There is also at least one hospital-based case-control histological study indicating that polymorphisms in XRCC4 may have an "influence" on prostate cancer susceptibility. Conditional (CD21-cre-mediated) deletion of the XRCC4 NHEJ gene in p53-deficient peripheral mouse B cells resulted in surface Ig-negative B-cell lymphomas, and these lymphomas often had a "reciprocal chromosomal translocation" fusing IgH to Myc (and also had "large chromosomal deletions or translocations" involving IgK or IgL, with IgL "fusing" to oncogenes or to IgH). XRCC4- and p53-deficient pro-B lymphomas "routinely activate c-myc by gene amplification"; and furthermore, it should be noted that XRCC4- and p53-deficient peripheral B-cell lymphomas "routinely ectopically activate" a single copy of c-myc. Indeed, in view of the observation by some that âDNA repair enzymes are correctives for DNA damage induced by carcinogens and anticancer drugsâ, it should not be surprising that âSNPs in DNA repair genes may play an important partâ in cancer susceptibility. In addition to the cancers identified above, XRCC4 polymorphisms have been identified as having a potential link to various additional cancers such as oral cancer, lung cancer, gastric cancer, and gliomas.
Based on the findings that (1) several polypeptides in the NHEJ pathway are "potential targets of autoantibodies" and (2) "one of the autoimmune epitopes in XRCC4 coincides with a sequence that is a nexus for radiation-induced regulatory events", it has been suggested that exposure to DNA double-strand break-introducing agents "may be one of the factors" mediating autoimmune responses.
There has been speculation that "XRCC4 codon 247*A and XRCC4 promoter -1394*T related genotypes and alleles . . . might be associated with higher endometriosis susceptibilities and pathogenesis".
Potential use as a cancer biomarker
In view of the possible associations of XRCC4 polymorphisms with risk of cancer susceptibility (see discussion above), XRCC4 could be used as a biomarker for cancer screening, particularly with respect to prostate cancer, breast cancer, and bladder cancer. In fact, XRCC4 polymorphisms were specifically identified as having the potential to be novel useful markers for "primary prevention and anticancer intervention" in the case of urothelial bladder cancer.
Radiosensitization of tumor cells
In view of the role of XRCC4 in DNA double-strand break repair, the relationship between impaired XRCC4 function and the radiosensitization of tumor cells has been investigated. For instance, it has been reported that "RNAi-mediated targeting of noncoding and coding sequences in DNA repair gene messages efficiently radiosensitizes human tumor cells".
Potential role in therapeutics
There has been discussion in the literature comcerning the potential role of XRCC4 in the development of novel therapeutics. For instance, Wu et al. have suggested that since the XRCC4 gene is "critical in NHEJ" and is "positively associated with cancer susceptibility", some XRCC4 SNPs such as G-1394T (rs6869366) "may serve as a common SNP for detecting and predict[ing] various cancers (so far for breast, gastric and prostate cancers . . .)"; and, although further investigation is needed, "they may serve as candidate targets for personalized anticancer drugs". The possibility of detecting endometriosis on this basis has also been mentioned, and this may also possibly lead to the eventual development of treatments. In evaluating further possibilites for anticancer treatments, Wu et al. also commented on the importance of âco-treatments of DNA-damaging agents and radiationâ. Specifically, Wu et al. noted that the âbalance between DNA damage and capacity of DNA repair mechanisms determines the final therapeutic outcomeâ and âthe capacity of cancer cells to complete DNA repair mechanisms is important for therapeutic resistance and has a negative impact upon therapeutic efficacyâ, and thus theorized that â[p]harmacological inhibition of recently detected targets of DNA repair with several small-molecule compounds . . . . has the potential to enhance the cytotoxicity of anticancer agentsâ.
Anti-XRCC4 antibodies include Alexa Fluor anti-XRCC4 mouse monoclonal antibody ab118008 (4H9), anti-XRCC4 rabbit polyclonal antibody ab157147 (N-terminal), and rabbit polyclonal anti-XRCC4 antibody ab145 (ChIP Grade) (all available from Abcam; Cambridge, MA, USA); phosphospecific antibodies to pS260 and pS318 in XRCC4, raised in sheep against the phosphopeptides: Ser260: SIISSLDVTD and Ser318: AENMSLETLR (phosphoserines underlined); and SAB2102728 (Sigma) anti-XRCC4 rabbit polyclonal antibody (available from Sigma-Aldrich; St. Louis, MO, USA). Antibodies to XRCC4 can have a variety of uses, including use in immunoassays to conduct research in areas such as DNA damage and repair, non-homologous end joining, transcription factors, epigenetics and nuclear signaling.
Research carried out in the 1980s revealed that a Chinese hamster ovary (CHO) cell mutant called XR-1 was "extremely sensitive" with regard to being killed by gamma rays during the G1 portion of the cell cycle but, in the same research studies, showed "nearly normal resistance" to gamma-ray damage during the late S phase; and in the course of this research, XR-1âs cell-cycle sensitivity was correlated with its inability to repair DNA double-strand breaks produced by ionizing radiation and restriction enzymes. In particular, in a study using somatic cell hybrids of XR-1 cells and human fibroblasts, Giaccia et al. (1989) showed that the XR-1 mutation was a recessive mutation; and in follow-up to this work, Giaccia et al. (1990) carried out further studies examining the XR-1 mutation (again using somatic cell hybrids formed between XR-1 and human fibroblasts) and were able to map the human complementing gene to chromosome 5 using chromosome-segregation analysis. Giaccia et al, tentatively assigned this human gene the name âXRCC4â (an abbreviation of âX-ray-complementing Chinese hamster gene 4â) and determined that (a) the newly-named XRCC4 gene biochemically restored the hamster defect to normal levels of resistance to gamma-ray radiation and bleomycin and (b) the XRCC4 gene restored the proficiency to repair DNA DSBs. Based on these findings, Giaccia et al. proposed that XRCC4 â as a single geneâ was responsible for the XR-1 phenotype.
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- Cifci S, Yilmaz M, Pehlivan M, Sever T, Okan V, Pehlivan S (November 2011). "DNA repair genes polymorphisms in multiple myeloma: no association with XRCC1 (Arg399Gln) polymorphism, but the XRCC4 (VNTR in intron 3 and G-1394T) and XPD (Lys751Gln) polymorphisms is associated with the disease in Turkish patients". Hematology 16 (6): 361â7. doi:10.1179/102453311X13127324303399. PMID 22183071.
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- Hsieh YY, Bau DT, Chang CC, Tsai CH, Chen CP, Tsai FJ (May 2008). "XRCC4 codon 247*A and XRCC4 promoter -1394*T related genotypes but not XRCC4 intron 3 gene polymorphism are associated with higher susceptibility for endometriosis". Mol. Reprod. Dev. 75 (5): 946â51. doi:10.1002/mrd.20829. PMID 18246529.
- Zheng Z, Ng WL, Zhang X, Olson JJ, Hao C, Curran WJ, Wang Y (March 2012). "RNAi-mediated targeting of noncoding and coding sequences in DNA repair gene messages efficiently radiosensitizes human tumor cells". Cancer Res. 72 (5): 1221â8. doi:10.1158/0008-5472.CAN-11-2785. PMID 22237628.
- Hsieh YY, Bau DT, Chang CC, Tsai CH, Chen CP, Tsai FJ (May 2008). "XRCC4 codon 247*A and XRCC4 promoter -1394*T related genotypes but not XRCC4 intron 3 gene polymorphism are associated with higher susceptibility for endometriosis". Mol. Reprod. Dev. 75 (5): 946â51. doi:10.1002/mrd.20829. PMID 18246529.
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- Giaccia AJ, Denko N, MacLaren R, Mirman D, Waldren C, Hart I, Stamato TD (September 1990). "Human chromosome 5 complements the DNA double-strand break-repair deficiency and gamma-ray sensitivity of the XR-1 hamster variant". Am. J. Hum. Genet. 47 (3): 459â69. PMC 1683886. PMID 1697445.
- Lieber MR (1999). "The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes". Genes Cells 4 (2): 77â85. doi:10.1046/j.1365-2443.1999.00245.x. PMID 10320474.
- Li Z, Otevrel T, Gao Y, Cheng HL, Seed B, Stamato TD, Taccioli GE, Alt FW (1996). "The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination". Cell 83 (7): 1079â89. doi:10.1016/0092-8674(95)90135-3. PMID 8548796.
- Grawunder U, Wilm M, Wu X, Kulesza P, Wilson TE, Mann M, Lieber MR (1997). "Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells". Nature 388 (6641): 492â5. doi:10.1038/41358. PMID 9242410.
- Critchlow SE, Bowater RP, Jackson SP (1997). "Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV". Curr. Biol. 7 (8): 588â98. doi:10.1016/S0960-9822(06)00258-2. PMID 9259561.
- Mizuta R, Cheng HL, Gao Y, Alt FW (1998). "Molecular genetic characterization of XRCC4 function". Int. Immunol. 9 (10): 1607â13. doi:10.1093/intimm/9.10.1607. PMID 9352367.
- Leber R, Wise TW, Mizuta R, Meek K (1998). "The XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase". J. Biol. Chem. 273 (3): 1794â801. doi:10.1074/jbc.273.3.1794. PMID 9430729.
- Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, Sekiguchi JM, Rathbun GA, Swat W, Wang J, Bronson RT, Malynn BA, Bryans M, Zhu C, Chaudhuri J, Davidson L, Ferrini R, Stamato T, Orkin SH, Greenberg ME, Alt FW (1999). "A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis". Cell 95 (7): 891â902. doi:10.1016/S0092-8674(00)81714-6. PMID 9875844.
- Modesti M, Hesse JE, Gellert M (1999). "DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity". EMBO J. 18 (7): 2008â18. doi:10.1093/emboj/18.7.2008. PMC 1171285. PMID 10202163.
- Nick McElhinny SA, Snowden CM, McCarville J, Ramsden DA (2000). "Ku recruits the XRCC4-ligase IV complex to DNA ends". Mol. Cell. Biol. 20 (9): 2996â3003. doi:10.1128/MCB.20.9.2996-3003.2000. PMC 85565. PMID 10757784.
- Gao Y, Ferguson DO, Xie W, Manis JP, Sekiguchi J, Frank KM, Chaudhuri J, Horner J, DePinho RA, Alt FW (2000). "Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development". Nature 404 (6780): 897â900. doi:10.1038/35009138. PMID 10786799.
- Chen L, Trujillo K, Sung P, Tomkinson AE (2000). "Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase". J. Biol. Chem. 275 (34): 26196â205. doi:10.1074/jbc.M000491200. PMID 10854421.
- Lee KJ, Huang J, Takeda Y, Dynan WS (2000). "DNA ligase IV and XRCC4 form a stable mixed tetramer that functions synergistically with other repair factors in a cell-free end-joining system". J. Biol. Chem. 275 (44): 34787â96. doi:10.1074/jbc.M004011200. PMID 10945980.
- Ford BN, Ruttan CC, Kyle VL, Brackley ME, Glickman BW (2000). "Identification of single nucleotide polymorphisms in human DNA repair genes". Carcinogenesis 21 (11): 1977â81. doi:10.1093/carcin/21.11.1977. PMID 11062157.
- Sibanda BL, Critchlow SE, Begun J, Pei XY, Jackson SP, Blundell TL, Pellegrini L (2002). "Crystal structure of an Xrcc4-DNA ligase IV complex". Nat. Struct. Biol. 8 (12): 1015â9. doi:10.1038/nsb725. PMID 11702069.
- Lee KJ, Dong X, Wang J, Takeda Y, Dynan WS (2002). "Identification of human autoantibodies to the DNA ligase IV/XRCC4 complex and mapping of an autoimmune epitope to a potential regulatory region". J. Immunol. 169 (6): 3413â21. PMID 12218164.
- Hsu HL, Yannone SM, Chen DJ (2003). "Defining interactions between DNA-PK and ligase IV/XRCC4". DNA Repair (Amst.) 1 (3): 225â35. doi:10.1016/S1568-7864(01)00018-0. PMID 12509254.
- XRCC4 protein, human at the US National Library of Medicine Medical Subject Headings (MeSH)
- FactorBook XRCC4
This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
DNA double-strand break repair and V(D)J recombination protein XRCC4 Provide feedback
This family consists of several eukaryotic DNA double-strand break repair and V(D)J recombination protein XRCC4 sequences. In the non-homologous end joining pathway of DNA double-strand break repair, the ligation step is catalysed by a complex of XRCC4 and DNA ligase IV. It is thought that XRCC4 and ligase IV are essential for alignment-based gap filling, as well as for final ligation of the breaks .
Lee JW, Yannone SM, Chen DJ, Povirk LF; , Cancer Res 2003;63:22-24.: Requirement for XRCC4 and DNA ligase IV in alignment-based gap filling for nonhomologous DNA end joining in vitro. PUBMED:12517771 EPMC:12517771
External database links
This tab holds annotation information from the InterPro database.
InterPro entry IPR010585
This entry represents the DNA double-strand break repair and V(D)J recombination protein XRCC4, which is found in certain Metazoans, fungi and plants. XRCC4 binds to DNA, and to DNA ligase IV (LIG4) to form the LIG4-XRCC4 complex [PUBMED:11029705]. The LIG4-XRCC4 complex is responsible for the ligation step in the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair. XRCC4 enhances the joining activity of LIG4. It is thought that XRCC4 and LIG4 are essential for alignment-based gap filling, as well as for final ligation of the breaks [PUBMED:12517771]. Binding of the LIG4-XRCC4 complex to DNA ends is dependent on the assembly of the DNA-dependent protein kinase complex DNA-PK to these DNA ends.
The mapping between Pfam and Gene Ontology is provided by InterPro. If you use this data please cite InterPro.
|Cellular component||nucleus (GO:0005634)|
|Molecular function||DNA binding (GO:0003677)|
|Biological process||double-strand break repair (GO:0006302)|
|DNA recombination (GO:0006310)|
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
The graphic that is shown by default represents the longest sequence with a given architecture. Each row contains the following information:
- the number of sequences which exhibit this architecture
a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one
Gladomain, followed by two consecutive
EGFdomains, and finally a single
- a link to the page in the Pfam site showing information about the sequence that the graphic describes
- the UniProt description of the protein sequence
- the number of residues in the sequence
- the Pfam graphic itself.
Note that you can see the family page for a particular domain by clicking on the graphic. You can also choose to see all sequences which have a given architecture by clicking on the Show link in each row.
Finally, because some families can be found in a very large number of architectures, we load only the first fifty architectures by default. If you want to see more architectures, click the button at the bottom of the page to load the next set.
Loading domain graphics...
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the NCBI sequence database, and our metagenomics sequence database. More...
There are various ways to view or download the sequence alignments that we store. We provide several sequence viewers and a plain-text Stockholm-format file for download.
We make a range of alignments for each Pfam-A family:
- the curated alignment from which the HMM for the family is built
- the alignment generated by searching the sequence database using the HMM
- Representative Proteomes (RPs) at 15%, 35%, 55% and 75% co-membership thresholds
- alignment generated by searching the NCBI sequence database using the family HMM
- alignment generated by searching the metagenomics sequence database using the family HMM
You can see the alignments as HTML or in three different sequence viewers:
- a Java applet developed at the University of Dundee. You will need Java installed before running jalview
- an HTML page showing the whole alignment.Please note: full Pfam alignments can be very large. These HTML views are extremely large and often cause problems for browsers. Please use either jalview or the Pfam viewer if you have trouble viewing the HTML version
- an HTML-based representation of the alignment, coloured according to the posterior-probability (PP) values from the HMM. As for the standard HTML view, heatmap alignments can also be very large and slow to render.
- Pfam viewer
- an HTML-based viewer that uses DAS to retrieve alignment fragments on request
You can download (or view in your browser) a text representation of a Pfam alignment in various formats:
You can also change the order in which sequences are listed in the alignment, change how insertions are represented, alter the characters that are used to represent gaps in sequences and, finally, choose whether to download the alignment or to view it in your browser directly.
You may find that large alignments cause problems for the viewers and the reformatting tool, so we also provide all alignments in Stockholm format. You can download either the plain text alignment, or a gzipped version of it.
We make a range of alignments for each Pfam-A family. You can see a description of each above. You can view these alignments in various ways but please note that some types of alignment are never generated while others may not be available for all families, most commonly because the alignments are too large to handle.
1Cannot generate PP/Heatmap alignments for seeds; no PP data available
Key: available, not generated, — not available.
Format an alignment
We make all of our alignments available in Stockholm format. You can download them here as raw, plain text files or as gzip-compressed files.
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
MyHits provides a collection of tools to handle multiple sequence alignments. For example, one can refine a seed alignment (sequence addition or removal, re-alignment or manual edition) and then search databases for remote homologs using HMMER3.
HMM logos is one way of visualising profile HMMs. Logos provide a quick overview of the properties of an HMM in a graphical form. You can see a more detailed description of HMM logos and find out how you can interpret them here. More...
If you find these logos useful in your own work, please consider citing the following article:
This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.
Note: You can also download the data file for the tree.
Curation and family details
This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.
|Seed source:||Pfam-B_21077 (release 10.0)|
|Number in seed:||5|
|Number in full:||165|
|Average length of the domain:||255.40 aa|
|Average identity of full alignment:||26 %|
|Average coverage of the sequence by the domain:||82.27 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 23193494 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||7|
|Download:||download the raw HMM for this family|
Weight segments by...
Change the size of the sunburst
selected sequences to HMM
a FASTA-format file
- 0 sequences
- 0 species
This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the More....
This chart is a modified "sunburst" visualisation of the species tree for this family. It shows each node in the tree as a separate arc, arranged radially with the superkingdoms at the centre and the species arrayed around the outermost ring.
How the sunburst is generated
The tree is built by considering the taxonomic lineage of each sequence that has a match to this family. For each node in the resulting tree, we draw an arc in the sunburst. The radius of the arc, its distance from the root node at the centre of the sunburst, shows the taxonomic level ("superkingdom", "kingdom", etc). The length of the arc represents either the number of sequences represented at a given level, or the number of species that are found beneath the node in the tree. The weighting scheme can be changed using the sunburst controls.
In order to reduce the complexity of the representation, we reduce the number of taxonomic levels that we show. We consider only the following eight major taxonomic levels:
Colouring and labels
Segments of the tree are coloured approximately according to their superkingdom. For example, archeal branches are coloured with shades of orange, eukaryotes in shades of purple, etc. The colour assignments are shown under the sunburst controls. Where space allows, the name of the taxonomic level will be written on the arc itself.
As you move your mouse across the sunburst, the current node will be highlighted. In the top section of the controls panel we show a summary of the lineage of the currently highlighed node. If you pause over an arc, a tooltip will be shown, giving the name of the taxonomic level in the title and a summary of the number of sequences and species below that node in the tree.
Anomalies in the taxonomy tree
There are some situations that the sunburst tree cannot easily handle and for which we have work-arounds in place.
Missing taxonomic levels
Some species in the taxonomic tree may not have one or more of the main eight levels that we display. For example, Bos taurus is not assigned an order in the NCBI taxonomic tree. In such cases we mark the omitted level with, for example, "No order", in both the tooltip and the lineage summary.
Unmapped species names
The tree is built by looking at each sequence in the full alignment for the family. We take the name of the species given by UniProt and try to map that to the full taxonomic tree from NCBI. In some cases, the name chosen by UniProt does not map to any node in the NCBI tree, perhaps because the chosen name is listed as a synonym or a misspelling in the NCBI taxonomy.
So that these nodes are not simply omitted from the sunburst tree, we group them together in a separate branch (or segment of the sunburst tree). Since we cannot determine the lineage for these unmapped species, we show all levels between the superkingdom and the species as "uncategorised".
Since we reduce the species tree to only the eight main taxonomic levels, sequences that are mapped to the sub-species level in the tree would not normally be shown. Rather than leave out these species, we map them instead to their parent species. So, for example, for sequences belonging to one of the Vibrio cholerae sub-species in the NCBI taxonomy, we show them instead as belonging to the species Vibrio cholerae.
Too many species/sequences
For large species trees, you may see blank regions in the outer layers of the sunburst. These occur when there are large numbers of arcs to be drawn in a small space. If an arc is less than approximately one pixel wide, it will not be drawn and the space will be left blank. You may still be able to get some information about the species in that region by moving your mouse across the area, but since each arc will be very small, it will be difficult to accurately locate a particular species.
The tree shows the occurrence of this domain across different species. More...
We show the species tree in one of two ways. For smaller trees we try to show an interactive representation, which allows you to select specific nodes in the tree and view them as an alignment or as a set of Pfam domain graphics.
Unfortunately we have found that there are problems viewing the interactive tree when the it becomes larger than a certain limit. Furthermore, we have found that Internet Explorer can become unresponsive when viewing some trees, regardless of their size. We therefore show a text representation of the species tree when the size is above a certain limit or if you are using Internet Explorer to view the site.
If you are using IE you can still load the interactive tree by clicking the "Generate interactive tree" button, but please be aware of the potential problems that the interactive species tree can cause.
For all of the domain matches in a full alignment, we count the number that are found on all sequences in the alignment. This total is shown in the purple box.
We also count the number of unique sequences on which each domain is found, which is shown in green. Note that a domain may appear multiple times on the same sequence, leading to the difference between these two numbers.
Finally, we group sequences from the same organism according to the NCBI code that is assigned by UniProt, allowing us to count the number of distinct sequences on which the domain is found. This value is shown in the pink boxes.
We use the NCBI species tree to group organisms according to their taxonomy and this forms the structure of the displayed tree. Note that in some cases the trees are too large (have too many nodes) to allow us to build an interactive tree, but in most cases you can still view the tree in a plain text, non-interactive representation. Those species which are represented in the seed alignment for this domain are highlighted.
You can use the tree controls to manipulate how the interactive tree is displayed:
- show/hide the summary boxes
- highlight species that are represented in the seed alignment
- expand/collapse the tree or expand it to a given depth
- select a sub-tree or a set of species within the tree and view them graphically or as an alignment
- save a plain text representation of the tree
Please note: for large trees this can take some time. While the tree is loading, you can safely switch away from this tab but if you browse away from the family page entirely, the tree will not be loaded.
For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the XRCC4 domain has been found. There are 30 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein seqence.
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