Resection-dependent canonical non-homologous end-joining induces genomic rearrangements

DNA double-strand breaks (DSBs) are the main threat to genomic integrity. The majority of DSBs are repaired by canonical non-homologous end-joining (c-NHEJ), where the two DSB ends are rejoined with minimal processing. Some studies suggest that the rejoining of DSBs by c-NHEJ can be error-prone by causing sequence alterations and/or the misrejoining of two separate DSBs. Such genomic rearrangements are a driving force in carcinogenesis. However, it remains an ongoing discussion as to how such rearrangements arise, as other studies associate genomic rearrangements with alternative end-joining (alt-EJ) and factors favoring resection. During alt-EJ the involvement of resection results in the loss of genetic information. However, there is evidence that alt-EJ mechanisms only play a role in human cells, which are deficient for certain repair proteins. This is the case in combination with genetic defects or in tumor cells. Thus, it remains unclear how mutagenic end-joining, which results in genomic rearrangements, operates and is regulated in wild-type human cells. To answer this question, mutagenic end-joining repair was investigated in this study by combining a reporter assay with other molecular biological assays. This reporter assay monitors the misrejoining of two 3.2 kilobase distant DSB ends under the loss of the intervening fragment. In addition, the sequence alterations at the misrejoined break sites were analyzed and overall repair was investigated. Furthermore, two approaches were taken to understand the circumstances under which error-prone end-joining is employed in wild-type human cells: the misrejoining of DSBs was compared between different reporter assays and the interactions of proteins involved in this pathway were analyzed. The results of this study show that distant DSB ends are misrejoined by a hitherto undescribed slow repair mechanism, which is specific for the G1 phase of the cell cycle. These misrejoined break sites are frequently associated with sequence alterations, especially small deletions (less than 50 nucleotides). These deletions are the result of limited resection. Surprisingly, the DSB ends remain protected by the key c-NHEJ factor KU during this undescribed repair mechanism. This feature distinguishes this end-joining process from all previously described pathways involving resection. Indeed, the absence of factors such as KU or the anti-resection factor 53BP1 results in increased genomic rearrangements by alt-EJ. Hence, in this novel repair pathway, c-NHEJ factors are pivotal in ensuring resection remains limited. Resection-dependent c-NHEJ is initiated by the activation of the resection factor CtIP. This is achieved through damage-inducible phosphorylation by the kinase PLK3 at Ser327, Thr847, and probably additional sites. Subsequently, the phosphorylation of CtIP at Ser327 results in its interaction with the pro-resection factor BRCA1. Similar to its role in S/G2 phase, the BRCA1-CtIP interaction seems to be important to overcome the resection barrier posed by 53BP1. Resection is executed by the exonuclease EXO1. Although EXD2 and MRE11 are also involved, the endonuclease function of MRE11 is dispensable for this repair pathway, indicating that resection is conducted from the DSB end. This is a key step limiting resection since the endonucleolytic cut by MRE11 overcomes the protected or blocked DSB ends during other resection processes to generate large single-stranded DNA overhangs. Thus, the initiation and execution of resection in this novel pathway is finely tuned to ensure that resection remains limited. Strikingly, the impairment of factors involved in the initiation or execution of resection does not result in unrepaired DSBs. This indicates that a repair pathway switch occurs in their absence. Although the absence of this novel error-prone repair pathway results in less genomic rearrangements, the remaining rearrangements are associated with worse sequence alterations, including longer deletions. Importantly, the limited resection process during resection-dependent c-NHEJ produces short single-stranded DNA regions, which form intermediate structures. These structures need to be resolved by the endonuclease activity of ARTEMIS, which is dependent on its interaction with the key c-NHEJ component DNA-PKcs. Thus, once resection has taken place, both ARTEMIS and DNA-PKcs are indispensable for repair completion. The break sites are mostly misrejoined using microhomologies, and thus require the single-stranded DNA gaps to be filled-in by polymerases. POL, which is typically associated with microhomologies, acts in this novel repair pathway, but the c-NHEJ-associated polymerases POL/ are also involved. The ligation step is conducted by the c-NHEJ ligase LIG4 and not by LIG1/3, which ligate DSB ends during alt-EJ. To summarize, despite its error-prone characteristics, this novel repair pathway restricts the loss of genetic information to a minimum. This is accomplished by rejoining the break sites using microhomologies, by keeping resection limited with the help of c-NHEJ factors, and by preventing an endonucleolytic cut by MRE11. In conclusion, this study characterizes a novel G1-specific mutagenic resection-dependent c-NHEJ pathway in human cells. In addition, this study shows how protein-protein interactions influence the choice to utilize this DSB repair pathway. The occurrence of resection in combination with c-NHEJ factors is a unique feature of this repair pathway and was hitherto thought to be mutually exclusive. Furthermore, the discovery of this pathway clarifies the role of alt-EJ as a type of backup mechanisms to compensate for missing repair proteins. Hence, in the ongoing discussion of how genomic rearrangements arise, this novel repair pathway unifies the contradicting observations of other studies

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847

DNA double-strand break resection occurs during non-homologous end joining in G1 but is distinct from resection during homologous recombination

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis

Resection-dependent canonical non-homologous end-joining induces genomic rearrangements

DNA double-strand breaks (DSBs) are the main threat to genomic integrity. The majority of DSBs are repaired by canonical non-homologous end-joining (c-NHEJ), where the two DSB ends are rejoined with minimal processing. Some studies suggest that the rejoining of DSBs by c-NHEJ can be error-prone by causing sequence alterations and/or the misrejoining of two separate DSBs. Such genomic rearrangements are a driving force in carcinogenesis. However, it remains an ongoing discussion as to how such rearrangements arise, as other studies associate genomic rearrangements with alternative end-joining (alt-EJ) and factors favoring resection. During alt-EJ the involvement of resection results in the loss of genetic information. However, there is evidence that alt-EJ mechanisms only play a role in human cells, which are deficient for certain repair proteins. This is the case in combination with genetic defects or in tumor cells. Thus, it remains unclear how mutagenic end-joining, which results in genomic rearrangements, operates and is regulated in wild-type human cells. To answer this question, mutagenic end-joining repair was investigated in this study by combining a reporter assay with other molecular biological assays. This reporter assay monitors the misrejoining of two 3.2 kilobase distant DSB ends under the loss of the intervening fragment. In addition, the sequence alterations at the misrejoined break sites were analyzed and overall repair was investigated. Furthermore, two approaches were taken to understand the circumstances under which error-prone end-joining is employed in wild-type human cells: the misrejoining of DSBs was compared between different reporter assays and the interactions of proteins involved in this pathway were analyzed. The results of this study show that distant DSB ends are misrejoined by a hitherto undescribed slow repair mechanism, which is specific for the G1 phase of the cell cycle. These misrejoined break sites are frequently associated with sequence alterations, especially small deletions (less than 50 nucleotides). These deletions are the result of limited resection. Surprisingly, the DSB ends remain protected by the key c-NHEJ factor KU during this undescribed repair mechanism. This feature distinguishes this end-joining process from all previously described pathways involving resection. Indeed, the absence of factors such as KU or the anti-resection factor 53BP1 results in increased genomic rearrangements by alt-EJ. Hence, in this novel repair pathway, c-NHEJ factors are pivotal in ensuring resection remains limited. Resection-dependent c-NHEJ is initiated by the activation of the resection factor CtIP. This is achieved through damage-inducible phosphorylation by the kinase PLK3 at Ser327, Thr847, and probably additional sites. Subsequently, the phosphorylation of CtIP at Ser327 results in its interaction with the pro-resection factor BRCA1. Similar to its role in S/G2 phase, the BRCA1-CtIP interaction seems to be important to overcome the resection barrier posed by 53BP1. Resection is executed by the exonuclease EXO1. Although EXD2 and MRE11 are also involved, the endonuclease function of MRE11 is dispensable for this repair pathway, indicating that resection is conducted from the DSB end. This is a key step limiting resection since the endonucleolytic cut by MRE11 overcomes the protected or blocked DSB ends during other resection processes to generate large single-stranded DNA overhangs. Thus, the initiation and execution of resection in this novel pathway is finely tuned to ensure that resection remains limited. Strikingly, the impairment of factors involved in the initiation or execution of resection does not result in unrepaired DSBs. This indicates that a repair pathway switch occurs in their absence. Although the absence of this novel error-prone repair pathway results in less genomic rearrangements, the remaining rearrangements are associated with worse sequence alterations, including longer deletions. Importantly, the limited resection process during resection-dependent c-NHEJ produces short single-stranded DNA regions, which form intermediate structures. These structures need to be resolved by the endonuclease activity of ARTEMIS, which is dependent on its interaction with the key c-NHEJ component DNA-PKcs. Thus, once resection has taken place, both ARTEMIS and DNA-PKcs are indispensable for repair completion. The break sites are mostly misrejoined using microhomologies, and thus require the single-stranded DNA gaps to be filled-in by polymerases. POL, which is typically associated with microhomologies, acts in this novel repair pathway, but the c-NHEJ-associated polymerases POL/ are also involved. The ligation step is conducted by the c-NHEJ ligase LIG4 and not by LIG1/3, which ligate DSB ends during alt-EJ. To summarize, despite its error-prone characteristics, this novel repair pathway restricts the loss of genetic information to a minimum. This is accomplished by rejoining the break sites using microhomologies, by keeping resection limited with the help of c-NHEJ factors, and by preventing an endonucleolytic cut by MRE11. In conclusion, this study characterizes a novel G1-specific mutagenic resection-dependent c-NHEJ pathway in human cells. In addition, this study shows how protein-protein interactions influence the choice to utilize this DSB repair pathway. The occurrence of resection in combination with c-NHEJ factors is a unique feature of this repair pathway and was hitherto thought to be mutually exclusive. Furthermore, the discovery of this pathway clarifies the role of alt-EJ as a type of backup mechanisms to compensate for missing repair proteins. Hence, in the ongoing discussion of how genomic rearrangements arise, this novel repair pathway unifies the contradicting observations of other studies

DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis