XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

Gu, Jiafeng; Lu, Haihui; Tippin, Brigette L.; Shimazaki, Noriko; Goodman, Myron F.; Lieber, Michael R.

doi:10.1038/sj.emboj.7601559

Cited by 140 publications

(139 citation statements)

References 49 publications

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“…Although their joining efficiencies differ, defects in KU80 or in XRCC4 both abrogated error-free end-joining. In non-fully complementary ends, wild-type cells primarily use the annealing of two of the 4Pnt, in a process consistent with in vitro biochemical data (25,26). PolX polymerases participate in the synthesis of nucleotide gaps (27,28).…”

Section: Discussionmentioning

confidence: 72%

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Guirouilh‐Barbat

Rass

Plo

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

152

183

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XRCC4-null mice have a more severe phenotype than KU80-null mice. Here, we address whether this difference in phenotype is connected to nonhomologous end-joining (NHEJ). We used intrachromosomal substrates to monitor NHEJ of two distal doublestrand breaks (DSBs) targeted by I-SceI, in living cells. In xrcc4-defective XR-1 cells, a residual but significant end-joining process exists, which primarily uses microhomologies distal from the DSB. However, NHEJ efficiency was strongly reduced in xrcc4-defective XR-1 cells versus complemented cells, contrasting with KU-deficient xrs6 cells, which showed levels of end-joining similar to those of complemented cells. Nevertheless, sequence analysis of the repair junctions indicated that the accuracy of end-joining was strongly affected in both xrcc4-deficient and KU-deficient cells. More specifically, these data showed that the KU80/XRCC4 pathway is conservative and not intrinsically error-prone but can accommodate non-fully complementary ends at the cost of limited mutagenesis.double-strand break repair ͉ genome rearrangements D NA double-strand breaks (DSBs) are harmful lesions generated by a variety of endogenous or exogenous stresses, potentially leading to genomic rearrangement. Nonhomologous endjoining (NHEJ) is a prominent pathway for DSB repair (1). Canonical NHEJ involves the successive intervention of the KU80-KU70 heterodimer, DNA-PKcs-Artemis, and, finally, ligase IV (Lig4) associated with its cofactors XRCC4 and Cernunnos/Xlf (2, 3). KU-independent NHEJ (KU-alt) has been described in vitro in both acellular extracts and cultured cells (1, 4-6).Alternative, XRCC4-independent DSB repair pathways (XRCC4-alt) have also been described, using episomic plasmid in cultured cells, using pulse field gel electrophoresis, or in in vitro biochemical experiments (5-10). One hypothesis could propose that XRCC4 and KU are implicated in the same canonical NHEJ pathway, whereas the alternate pathway is independent of both KU and XRCC4. However, in transgenic mice, the inactivation of XRCC4 or Lig4 results in a more severe phenotype than the inactivation of KU (11), suggesting that XRCC4 might have an additional essential function; however, studies show that XRCC4 and Lig4 do not have roles outside of NHEJ, whereas in contrast, KU acts in other processes such as transcription, apoptosis, and responses to the cell microenvironment (12)(13)(14).Alternatively, these varying phenotypes in mice may actually result from differences in DSB repair efficiencies, indicating that defects in XRCC4 might be more deleterious for DSB repair than defects in KU. What challenges this hypothesis, however, is that substantial class switch recombination (CSR) has recently been shown to occur in mouse B cells without XRCC4, whereas no CSR was recorded in cells devoid of KU (15-18).The relative contributions of XRCC4 and KU80 versus the XRCC4-alt and KU-alt pathways, respectively, to DSB repair remain unclear in wild-type cells. Contrasting results were obtained in living cells, using an episomic plasm...

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Section: Discussionmentioning

confidence: 72%

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Guirouilh‐Barbat

Rass

Plo

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

152

183

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“…These proteins are structurally similar to and interact with the yeast Lif1 and human XRCC4 subunit of the DNA ligase IV complex (20 -23, 25, 28, 30, 35). In addition, XLF enhances joining by DNA ligase IV-XRCC4 of both matched and mismatched DNA ends (31)(32)(33)(34)46). Using a combination of molecular genetic and biochemical approaches, we have demonstrated that Nej1 not only contributes to the final ligation step of NHEJ but also plays a critical role in the initiation of the NHEJ pathway.…”

Section: Discussionmentioning

confidence: 99%

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

Chen¹,

Tomkinson

2011

Journal of Biological Chemistry

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In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway.

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“…Although DSBs are detectable in B cells with AID +/C-termΔ mutations, they are undetectable in UNG- deficient B cells (4,20). Fewer AID-and/or UNG-induced DNA lesions likely leads to staggered and long single-stranded DNA breaks that may require extensive processing by polymerases, such as polymerase μ or ζ (34,35). Polymerase-mediated repair might lead to the insertion of nucleotides at the junctions due to template-independent synthesis, or to microhomologies due to fillin synthesis (34,36).…”

Section: Position Of Sμ and Sα Breakpoints In Switchmentioning

confidence: 99%

“…Fewer AID-and/or UNG-induced DNA lesions likely leads to staggered and long single-stranded DNA breaks that may require extensive processing by polymerases, such as polymerase μ or ζ (34,35). Polymerase-mediated repair might lead to the insertion of nucleotides at the junctions due to template-independent synthesis, or to microhomologies due to fillin synthesis (34,36). The polymerase-mediated repair is likely associated with the components of c-NHEJ (34); however, the microhomology length of >10 nucleotides and the location of the Sμ breakpoints observed in the analyzed patients argues for another pathway involving DNA end-resection to search for donor/acceptor homology.…”

Section: Position Of Sμ and Sα Breakpoints In Switchmentioning

confidence: 99%

Impaired induction of DNA lesions during immunoglobulin class-switch recombination in humans influences end-joining repair

Kracker

Imai

Gardès

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Ig class-switch recombination (CSR) is a region-specific process that exchanges the constant Ig heavy-chain region and thus modifies an antibody's effector function. DNA lesions in switch (S) regions are induced by activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase 2 (UNG2), subsequently processed to DNA breaks, and resolved by either the classical nonhomologous endjoining pathway or the alternative end-joining pathway (XRCC4/ DNA ligase 4-and/or Ku70/Ku80-independent and prone to increased microhomology usage). We examined whether the induction of DNA lesions influences DNA end-joining during CSR by analyzing Sμ-Sα recombination junctions in various human Ig CSR defects of DNA lesion induction. We observed a progressive trend toward the usage of microhomology in Sμ-Sα recombination junctions from AID-heterozygous to AID-autosomal dominant to UNG2-deficient B lymphocytes. We thus hypothesize that impaired induction of DNA lesions in S regions during CSR leads to unusual end-processing of the DNA breaks, resulting in microhomology-mediated end-joining, which could be an indication for preferential processing by alternative end-joining rather than by classical nonhomologous end-joining.antibody maturation | switch junction | DNA repair D uring immune responses, mature B cells diversify their Ig genes through class-switch recombination (CSR) and somatic hypermutation (SHM). The latter mechanism introduces nontemplated point mutations in the variable region of Ig genes and thereby enables the selection of antibodies with increased affinity for the antigen. CSR modulates antibody effector function by replacing one constant region with another in a deletionmediated recombination process, while retaining the binding specificity (variable region) of the B-cell receptor. Both processes are initiated by the enzyme activation-induced cytidine deaminase (AID), which deaminates cytosine to produce U:G mismatches in target DNA (1-3). GC-rich regions, so-called "switch" (S) regions, present in front of each constant region and of varying lengths, are the target DNAs for AID during CSR. The main route for processing AID-induced lesions involves uracil excision through several pathways, primarily the pathway involving uracil-DNA glycosylase (UNG2) (4, 5). Abasic sites are further processed to generate DNA double-strand breaks (DSBs), which are obligate intermediates in CSR (6, 7). The DSBs activate damage response proteins, such as PI3-like protein kinase ataxia-telangiectasia mutated, the phosphorylated histone variant H2AX, the MRN complex (MRE11, RAD50, and NBS1), MDC1, and 53BP1, all of which are known to play roles in CSR in promoting appropriate repair and efficient long-range, region-specific recombination (8)(9)(10)(11)(12)(13)(14). In CSR, the resolution step is normally mediated by the classical nonhomologous end-joining pathway (c-NHEJ); however, recent reports strongly suggest that resolution also can be mediated (albeit at a lower frequency) by Ku 70-, Ku 80-, and/or XRCC4/DNA ligase 4-independen...

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XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

Cited by 140 publications

References 49 publications

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

Impaired induction of DNA lesions during immunoglobulin class-switch recombination in humans influences end-joining repair

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