Involvement of Polynucleotide Kinase in a Poly(ADP-ribose) Polymerase-1-dependent DNA Double-strand Breaks Rejoining Pathway

Audebert, Marc; Salles, Bernard; Weinfeld, Michael; Calsou, Patrick

doi:10.1016/j.jmb.2005.11.028

Cited by 93 publications

(65 citation statements)

References 52 publications

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“…Consistent with this, PARP1 has been found to facilitate synapsis of double-strand breaks, and can mediate a Ku-independent mechanism of microhomology end joining [41][42][43] .…”

Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 54%

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks

Ali¹,

Timinszky²,

Arribas-Bosacoma³

et al. 2012

Nat Struct Mol Biol

225

196

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Poly(ADP-ribose) polymerase I (PARP1) is a primary DNA damage sensor whose (ADP-ribose) polymerase activity is acutely regulated by interaction with DNA breaks. Upon activation at sites of DNA damage, PARP1 modifies itself and other proteins by covalent addition of long branched polymers of ADP-ribose, which in turn recruit downstream DNA repair and chromatin remodelling factors. PARP1 recognizes DNA damage through its N-terminal DNA-binding domain (DBD), which consists of a tandem repeat of an unusual zinc-finger (ZnF) domain. We have now determined the crystal structure of the human PARP1-DBD bound to a DNA break. Along with functional analysis of PARP1 recruitment to sites of DNA damage in vivo, the structure reveals a dimeric assembly whereby ZnF1 and ZnF2 domains from separate PARP1 molecules form a strand-break recognition module that helps activate PARP1 by facilitating its dimerization and consequent trans-automodification.Short-patch repair of DNA single-strand breaks is initiated by poly(ADP-ribose) polymerase-1 (PARP1) -a multi-domain enzyme activated by binding of its N-terminal DNA-binding domain (DBD) to DNA breaks [1][2][3][4][5] . Activated PARP1 utilises NAD + to Correspondence to: Andreas G. Ladurner; Laurence H. Pearl; Antony W. Oliver. AUTHOR CONTRIBUTIONS A.A.E.A. purified the protein, crystallized the complex and collected the X-ray diffraction data; G.T. designed and constructed the PARP1-EGFP constructs and performed the laser DNA damage experiments; M.K. performed the FRAP experiments; P.O.H. engineered the knockdown PARP1 cell line and the wild-type imaging reporter constructs, and performed the in vitro complementation assays; M.H. and R.A.-B. engineered and purified mutant PARP1 constructs; A.G.L. designed the study and analysed the data; L.H.P designed the study, analysed the data and wrote the paper; A.W.O. made the baculovirus constructs, designed the purification protocol, and solved and refined the crystal structure. All authors discussed the results and commented on the manuscript. We have now determined the crystal structure of the DNA-binding domain of PARP1 (PARP1-DBD) encompassing the first two zinc-finger (ZnF) domains, bound to a DNA break. In contrast with structural analysis of the separated domains 22 , we show that DNA binding by both zinc-finger domains is essential to damage recruitment in vivo, and that ZnF1 and ZnF2 domains from separate PARP1 molecules act as a functional unit to generate a dimeric binding module that specifically recognizes the single-strand / double-strand transition at a recessed DNA break. Mutational analysis in vitro and in cells demonstrates the functional requirement for zinc-finger dimerisation and reveals a mechanism for bringing two PARP1 molecules into close proximity at a DNA break as a prerequisite for transmodification. RESULTS Structure of the PARP1-DBD -DNA ComplexAn N-terminal segment of human PARP1 (residues 5-202) was expressed in insect cells and purified by column chromatography. Screening with a range of DNA molecules...

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“…Consistent with this, PARP1 has been found to facilitate synapsis of double-strand breaks, and can mediate a Ku-independent mechanism of microhomology end joining [41][42][43] .…”

Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 54%

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks

Ali¹,

Timinszky²,

Arribas-Bosacoma³

et al. 2012

Nat Struct Mol Biol

225

196

View full text Add to dashboard Cite

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“…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)(6)(7)(8)(9)(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.…”

mentioning

confidence: 99%

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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“…Genetic studies have established a role for PARP-1 in HR (Hochegger et al, 2006;Idogawa et al, 2007), whereas biochemical and genetic studies have linked PARP-1 to NHEJ (Schultz et al, 2003;Perrault et al, 2004;Audebert et al, 2006;Wang et al, 2006). PARP-1 has been shown to interact with the NHEJ proteins, Ku antigen and DNA-dependent protein kinase (DNA-PK) with reciprocal modifications and mutual alterations in activity suggested (Ruscetti et al, 1998;Ariumi et al, 1999;Galande and KohwiShigematsu, 1999).…”

Section: Introductionmentioning

confidence: 99%

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

Fang¹,

Soubeyrand²,

Haché

2010

Oncogene

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Poly (ADP-ribose) polymerase-1 (PARP-1) has an important role in the cellular response to a broad spectrum of DNA lesions. PARP-1 is strongly activated in response to double-stranded DNA breaks (DSBs), yet its contribution to the DSB response is poorly understood. Here we used bleomycin, a radiomimetic that generates DSBs with high specificity to focus on the response of PARP-1 to DSBs. We report that the induction of PARP-1 activity by bleomycin depends on the Ku antigen, a nonhomologous-DNA-End-Joining factor and protein phosphatase 5 (PP5). PARP-1 activation in response to bleomycin was reduced over 10-fold in Ku-deficient cells, whereas its activation in response to U.V. was unaffected. PARP-1 activation was rescued by reexpression of Ku, but was refractory to manipulation of DNA-dependent protein kinase or ATM. Similarly, PARP-1 activation subsequent to bleomycin was reduced 2-fold on ablation of PP5 and was increased 5-fold when PP5 was overexpressed. PP5 seemed to act directly on PARP-1, as its basal phosphorylation was reduced on overexpression of PP5, and PP5 dephosphorylated PARP-1 in vitro. These results highlight the functional importance of Ku antigen and PP5 for PARP-1 activity subsequent to DSBs.

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Involvement of Polynucleotide Kinase in a Poly(ADP-ribose) Polymerase-1-dependent DNA Double-strand Breaks Rejoining Pathway

Cited by 93 publications

References 52 publications

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks

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

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

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