Summary Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase α, and PCNA. The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a novel DNA binding architecture composed of an OB-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, pol α, and DNA interactions onto the crystal structure provides insight into how Mcm10 may coordinate protein and DNA binding within the replisome.

Helicase-like transcription factor (HLTF) is a central mediator of the DNA damage response and maintains genome stability by regressing stalled replication forks. The N-terminal HIRAN domain binds specifically to the 3'-end of single-stranded DNA (ssDNA), and disrupting this function interferes with fork regression as well as replication fork progression in cells under replication stress. Here, we investigated the mechanism by which the HIRAN-ssDNA interaction facilitates fork remodeling. Our results indicated that HIRAN capture of a denatured nascent leading 3'-end directs specific binding of HLTF to forks. DNase footprinting revealed that HLTF binds to the parental duplex ahead of the fork and at the leading edge behind the fork. Moreover, we found that the HIRAN domain is important for initiating regression of forks when both nascent strands are at the junction, but is dispensable when forks contain ssDNA regions on either template strand. We also found that HLTF catalyzes fork restoration from a partially regressed structure in a HIRAN-dependent manner. Thus, HIRAN serves as a substrate-recognition domain to properly orient the ATPase motor domain at stalled and regressed forks and initiates fork remodeling by guiding formation of a four-way junction. We discuss how these activities compare with those of two related fork remodelers, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) and zinc finger RANBP2 type-containing 3 (ZRANB3) to provide insight into their nonredundant roles in DNA damage tolerance.

SMARCAL1, a DNA remodeling protein fundamental to genome integrity during replication, is the only gene associated with the developmental disorder Schimke immuno-osseous dysplasia (SIOD). SMARCAL1-deficient cells show collapsed replication forks, S-phase cell cycle arrest, increased chromosomal breaks, hypersensitivity to genotoxic agents, and chromosomal instability. The SMARCAL1 catalytic domain (SMARCAL1 CD ) is composed of an SNF2-type doublestranded DNA motor ATPase fused to a HARP domain of unknown function. The mechanisms by which SMARCAL1 and other DNA translocases repair replication forks are poorly understood, in part because of a lack of structural information on the domains outside of the common ATPase motor. In the present work, we determined the crystal structure of the SMARCAL1 HARP domain and examined its conformation and assembly in solution by small angle X-ray scattering. We report that this domain is conserved with the DNA mismatch and damage recognition domains of MutS/MSH and NER helicase XPB, respectively, as well as with the putative DNA specificity motif of the T4 phage fork regression protein UvsW. Loss of UvsW fork regression activity by deletion of this domain was rescued by its replacement with HARP, establishing the importance of this domain in UvsW and demonstrating a functional complementarity between these structurally homologous domains. Mutation of predicted DNA-binding residues in HARP dramatically reduced fork binding and regression activities of SMARCAL1 CD . Thus, this work has uncovered a conserved substrate recognition domain in DNA repair enzymes that couples ATP-hydrolysis to remodeling of a variety of DNA structures, and provides insight into this domain's role in replication fork stability and genome integrity.replication restart | fork reversal A ccurate DNA replication is essential for genomic stability (1). Aberrant DNA structures, protein barriers, and chemically modified DNA thwart progression of the replication fork and lead to mutations, cytotoxicity, and increased chromosomal rearrangements (2-5). Uncoupling of polymerase and helicase activities at a stalled fork (6) results in an accumulation of singlestranded (ss) DNA (Fig. 1A), rendering the genome susceptible to nuclease cleavage and thereby increasing the probability of genetic rearrangements (7-9). Failure to stabilize stalled forks can lead to replisome dissociation and fork degradation or collapse. DNA damage response (DDR) pathways maintain genomic integrity by rectifying or protecting stalled or collapsed forks and regulating DNA repair (9-11). In humans, accumulation of the ssDNA-binding protein Replication Protein A (RPA) at stalled forks signals recruitment of the S-phase checkpoint kinase ATR (12-15), consequently triggering recruitment of proteins that promote sister chromatid cohesion and stabilize the histone/chromatin structure (16).Stabilization, repair, and restart of stalled forks can involve regression of the fork into a four-stranded "chicken foot" structure, in which the nascent DNA str...

The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.

Background: Integrin ␣1␤1 binding to collagen IV down-regulates collagen synthesis, but how modulating integrin ␣1␤1 affinity alters collagen homeostasis is unknown. Results: Cells carrying mutations of the ␣1 subunit with enhanced collagen binding show increased ERK activation and increased collagen down-regulation. Conclusion: Enhancing the affinity of the ␣1 subunit to collagen potentiates integrin ␣1␤1-mediated outside-in signaling. Significance: These findings have major implications for pathological conditions such as fibrotic diseases.

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Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress in Xenopus egg extracts. We found that the helicase RTEL1 and the replisome protein MCM10 were highly enriched on DNA under these conditions. We show that RTEL1 normally plays a minor role during fork convergence while the role of MCM10 is normally negligible. However, RTEL1 and MCM10 both become crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 exert non-additive effects on fork convergence and physically interact, suggesting that they function together. Furthermore, RTEL1 and MCM10 do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 appear to play a general role in promoting progression of stalled forks, including when forks stall during termination. Overall, our data identify an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.