ATRX Promotes DNA Repair Synthesis and Sister Chromatid Exchange during Homologous Recombination.

ATRX is a chromatin remodeler that, together with its chaperone DAXX, deposits the histone variant H3.3 in pericentromeric and telomeric regions. Notably, ATRX is frequently mutated in tumors that maintain telomere length by a specific form of homologous recombination (HR). Surprisingly, in this context, we demonstrate that ATRX-deficient cells exhibit a defect in repairing exogenously induced DNA double-strand breaks (DSBs) by HR. ATRX operates downstream of the Rad51 removal step and interacts with PCNA and RFC-1, which are collectively required for DNA repair synthesis during HR. ATRX depletion abolishes DNA repair synthesis and prevents the formation of sister chromatid exchanges at exogenously induced DSBs. DAXX- and H3.3-depleted cells exhibit identical HR defects as ATRX-depleted cells, and both ATRX and DAXX function to deposit H3.3 during DNA repair synthesis. This suggests that ATRX facilitates the chromatin reconstitution required for extended DNA repair synthesis and sister chromatid exchange during HR.

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.

ATRX and RECQ5 define distinct homologous recombination subpathways.

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

Poly(ADP-ribose)-dependent chromatin unfolding facilitates the association of DNA-binding proteins with DNA at sites of damage.

The addition of poly(ADP-ribose) (PAR) chains along the chromatin fiber due to PARP1 activity regulates the recruitment of multiple factors to sites of DNA damage. In this manuscript, we investigated how, besides direct binding to PAR, early chromatin unfolding events controlled by PAR signaling contribute to recruitment to DNA lesions. We observed that different DNA-binding, but not histone-binding, domains accumulate at damaged chromatin in a PAR-dependent manner, and that this recruitment correlates with their affinity for DNA. Our findings indicate that this recruitment is promoted by early PAR-dependent chromatin remodeling rather than direct interaction with PAR. Moreover, recruitment is not the consequence of reduced molecular crowding at unfolded damaged chromatin but instead originates from facilitated binding to more exposed DNA. These findings are further substantiated by the observation that PAR-dependent chromatin remodeling at DNA lesions underlies increased DNAse hypersensitivity. Finally, the relevance of this new mode of PAR-dependent recruitment to DNA lesions is demonstrated by the observation that reducing the affinity for DNA of both CHD4 and HP1α, two proteins shown to be involved in the DNA-damage response, strongly impairs their recruitment to DNA lesions.

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling.

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.

Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance.

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.

Role of SUMO modification of human PCNA at stalled replication fork.

DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.

Protein Phosphatase 4 Is Required for Centrobin Function in DNA Damage Repair.

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein phosphatase 4 (PP4) plays a pivotal role in coordinating cellular response to DNA damage. Meanwhile, Centrobin (CNTRB), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with CNTRB in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and CNTRB and shed light on the importance of two specific motifs in CNTRB, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells depleted of PP4 or CNTRB and cells harboring FRVP and SQ mutations in CNTRB, which result in similar abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms of PP4 and CNTRB-regulated HR repair and their interrelation.

The chromatin remodeler ALC1 underlies resistance to PARP inhibitor treatment.

Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the treatment of BRCA-deficient cancers, with treatments currently extending toward other homologous recombination defective tumors. In a genome-wide CRISPR knockout screen with olaparib, we identify ALC1 (Amplified in Liver Cancer 1)-a cancer-relevant poly(ADP-ribose)-regulated chromatin remodeling enzyme-as a key modulator of sensitivity to PARP inhibitor. We found that ALC1 can remove inactive PARP1 indirectly through binding to PARylated chromatin. Consequently, ALC1 deficiency enhances trapping of inhibited PARP1, which then impairs the binding of both nonhomologous end-joining and homologous recombination repair factors to DNA lesions. We also establish that ALC1 overexpression, a common feature in multiple tumor types, reduces the sensitivity of BRCA-deficient cells to PARP inhibitors. Together, we conclude that ALC1-dependent PARP1 mobilization is a key step underlying PARP inhibitor resistance.

DNA repair synthesis and histone deposition partner during homologous recombination.

Chromatin remodeling is critical for the regulation of the DNA damage response. We highlight findings from our recent study showing that the deposition of the histone variant H3.3 by the alpha-thalassemia mental retardation X-linked protein (ATRX) and the death domain associated protein (DAXX) chromatin remodeling complex regulates DNA repair synthesis during homologous recombination.

The DNA-binding box of human SPARTAN contributes to the targeting of Polη to DNA damage sites.

Inappropriate repair of UV-induced DNA damage results in human diseases such as Xeroderma pigmentosum (XP), which is associated with an extremely high risk of skin cancer. A variant form of XP is caused by the absence of Polη, which is normally able to bypass UV-induced DNA lesions in an error-free manner. However, Polη is highly error prone when replicating undamaged DNA and, thus, the regulation of the proper targeting of Polη is crucial for the prevention of mutagenesis and UV-induced cancer formation. Spartan is a novel regulator of the damage tolerance pathway, and its association with Ub-PCNA has a role in Polη targeting; however, our knowledge about its function is only rudimentary. Here, we describe a new biochemical property of purified human SPARTAN by showing that it is a DNA-binding protein. Using a DNA binding mutant, we provide in vivo evidence that DNA binding by SPARTAN regulates the targeting of Polη to damage sites after UV exposure, and this function contributes highly to its DNA-damage tolerance function.

Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans.

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.