Multiple Functions for Mcm2–7 ATPase Motifs during Replication Initiation

The Mcm2–7 replicative helicase is central to all steps of eukaryotic DNA replication. The hexameric ring of Mcm subunits forms six essential ATPases whose contributions to replication initiation remain unclear. Mcm2–7 complexes containing ATPase-motif mutations showed Mcm2–7 ATP binding and hydrolysis are required for helicase loading. Loading-defective Mcm2–7 mutant complexes were defective in initial Mcm2–7 recruitment or Cdt1 release. Comparison with Cdc6 ATPase mutants showed that Cdc6 ATP hydrolysis is not required for helicase loading but instead drives removal of Mcm2–7 complexes that cannot complete loading. A subset of Mcm2–7 ATPase-site mutants completed helicase loading but could not initiate replication. Individual mutants were defective in distinct events during helicase activation, including maintenance of DNA association, recruitment of the GINS helicase activator, and DNA unwinding. Consistent with its heterohexameric structure, our findings show that the six Mcm2–7 ATPase active sites are specialized for different functions during helicase loading and activation.United States. National Institutes of Health (GM052339)United States. National Institutes of Health (GM007287)National Science Foundation (U.S.) (1122374

Dimeric configuration of SeqA protein bound to a pair of hemi-methylated GATC sequences

The binding of SeqA protein to hemi-methylated GATC sequences (hemi-sites) regulates chromosome initiation and the segregation of replicated chromosome in Escherichia coli. We have used atomic force microscopy to examine the architecture of SeqA and the mode of binding of one molecule of SeqA to a pair of hemi-sites in aqueous solution. SeqA has a bipartite structure composed of a large and a small lobe. Upon binding of a SeqA molecule to a pair of hemi-sites, the larger lobe becomes visibly separated into two DNA binding domains, each of which binds to one hemi-site. The two DNA binding domains are held together by association between the two multimerization domains that make up the smaller lobe. The binding of each DNA binding domain to a hemi-site leads to bending of the bound DNA inwards toward the bound protein. In this way, SeqA adopts a dimeric configuration when bound to a pair of hemi-sites. Mutational analysis of the multimerization domain indicates that, in addition to multimerization of SeqA polypeptides, this domain contributes to the ability of SeqA to bind to a pair of hemi-sites and to its cooperative behavior

ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors

Eukaryotic DNA replication: Orchestrated action of multi-subunit protein complexes

Genome duplication is an essential process to preserve genetic information between generations. The eukaryotic cell cycle is composed of functionally distinct phases: G1, S, G2, and M. One of the key replicative proteins that participate at every stage of DNA replication is the Mcm2-7 complex, a replicative helicase. In the G1 phase, inactive Mcm2-7 complexes are loaded on the replication origins by replication-initiator proteins, ORC and Cdc6. Two kinases, S-CDK and DDK, convert the inactive origin-loaded Mcm2-7 complex to an active helicase, the CMG complex in the S phase. The activated CMG complex begins DNA unwinding and recruits enzymes essential for DNA synthesis to assemble a replisome at the replication fork. After completion of DNA synthesis, the inactive CMG complex on the replicated DNA is removed from chromatin to terminate DNA replication. In this review, we will discuss the structure, function, and regulation of the molecular machines involved in each step of DNA replication

Single Molecule Measurements Reveal Conformational Transitions During DNA Clamp Loading and Unloading

Proliferation cell nuclear antigen (PCNA) is a DNA clamp, playing an important role of providing a ??????platform?????? for various enzymes during DNA replication. The loading of the closed trimeric ring of PCNA into duplex DNA requires the ATP-dependent activity of replication factor C (RFC) complex. The unloading of PCNA from chromatin is crucial for the regulation of replication process and maintaining genomic stability and it was recently found that ATAD5 protein is complexed with RFC-like complex (RLC) to get involved in the unloading of PCNA. However, the molecular mechanisms of PCNA loading and unloading processes have remained poorly understood. Here, we report direct observation of the loading and unloading dynamics of human PCNA driven by RFC and ATAD5-RLC complexes, respectively, by single molecule fluorescence resonance energy transfer measurements. Distinct conformational stages during PCNA loading were clearly detected that represent open and closed conformations of PCNA trimer and another loading intermediate that possibly triggers the dissociation of RFC complex. The unloading of PCNA occurred upon binding ATAD5-RLC complex through an intermediate conformation, but not requiring the hydrolysis of ATP, which is later employed to recycle PCNA and ATAD5-RLC complexes. Our findings present a mechanistic model of the clamp loading/unloading dynamics and bring implications on the mechanism controlling the exclusive functions of RFC and ATAD5-RLC complexes in the loading and unloading processes, respectively. It further provides a platform to study how PCNA controls the exchange of various replication enzymes

Replication origin-flanking roadblocks reveal origin-licensing dynamics and altered sequence dependence

In eukaryotes, DNA replication initiates from multiple origins of replication for timely genome duplication. These sites are selected by origin licensing, during which the core enzyme of the eukaryotic DNA replicative helicase, the Mcm2-7 (minichromosome maintenance) complex, is loaded at each origin. This origin licensing requires loading two Mcm2-7 helicases around origin DNA in a head-to-head orientation. Current models suggest that the origin-recognition complex (ORC) and cell-division cycle 6 (Cdc6) proteins recognize and encircle origin DNA and assemble an Mcm2-7 double-hexamer around adjacent double-stranded DNA. To test this model and assess the location of Mcm2-7 initial loading, we placed DNA-protein roadblocks at defined positions adjacent to the essential ORC-binding site within Saccharomyces cerevisiae origin DNA. Roadblocks were made either by covalent cross-linking of the HpaII methyltransferase to DNA or through binding of a transcription activator-like effector (TALE) protein. Contrary to the sites of Mcm2-7 recruitment being precisely defined, only single roadblocks that inhibited ORC-DNA binding showed helicase loading defects. We observed inhibition of helicase loading without inhibition of ORC-DNA binding only when roadblocks were placed on both sides of the origin to restrict sliding of a helicase-loading intermediate. Consistent with a sliding helicase-loading intermediate, when either one of the flanking roadblocks was eliminated, the remaining roadblock had no effect on helicase loading. Interestingly, either origin-flanking nucleosomes or roadblocks resulted in helicase loading being dependent on an additional origin sequence known to be a weaker ORC-DNA-binding site. Together, our findings support a model in which sliding helicase-loading intermediates increase the flexibility of the DNA sequence requirements for origin licensing111sciescopu

Replication origin–flanking roadblocks reveal origin-licensing dynamics and altered sequence dependence

In eukaryotes, DNA replication initiates from multiple origins of replication for timely genome duplication. These sites are selected by origin licensing, during which the core enzyme of the eukaryotic DNA replicative helicase, the Mcm2-7 (minichromosome maintenance) complex, is loaded at each origin. This origin licensing requires loading two Mcm2-7 helicases around origin DNA in a head-to-head orientation. Current models suggest that the origin-recognition complex (ORC) and cell-division cycle 6 (Cdc6) proteins recognize and encircle origin DNA and assemble an Mcm2-7 double-hexamer around adjacent double-stranded DNA. To test this model and assess the location of Mcm2-7 initial loading, we placed DNA-protein roadblocks at defined positions adjacent to the essential ORC-binding site within Saccharomyces cerevisiae origin DNA. Roadblocks were made either by covalent cross-linking of the HpaII methyltransferase to DNA or through binding of a transcription activator-like effector (TALE) protein. Contrary to the sites of Mcm2-7 recruitment being precisely defined, only single roadblocks that inhibited ORC-DNA binding showed helicase loading defects. We observed inhibition of helicase loading without inhibition of ORC-DNA binding only when roadblocks were placed on both sides of the origin to restrict sliding of a helicase-loading intermediate. Consistent with a sliding helicase-loading intermediate, when either one of the flanking roadblocks was eliminated, the remaining roadblock had no effect on helicase loading. Interestingly, either origin-flanking nucleosomes or roadblocks resulted in helicase loading being dependent on an additional origin sequence known to be a weaker ORC-DNA-binding site. Together, our findings support a model in which sliding helicase-loading intermediates increase the flexibility of the DNA sequence requirements for origin licensing.American Cancer Society. Postdoctoral Fellowship (123700-PF-13-071-01-DMC)National Institutes of Health (U.S.). Pre-Doctoral Training Program (Grant GM007287)National Science Foundation (U.S.). Graduate Research Fellowship (1122374)National Cancer Institute (U.S.) (Grant P30-CA14051

Distinct Motifs in ATAD5 C-Terminal Domain Modulate PCNA Unloading Process

Proliferating cell nuclear antigen (PCNA) is a DNA clamp that functions in key roles for DNA replication and repair. After the completion of DNA synthesis, PCNA should be unloaded from DNA in a timely way. The ATAD5-RFC-Like Complex (ATAD5-RLC) unloads PCNA from DNA. However, the mechanism of the PCNA-unloading process remains unclear. In this study, we determined the minimal PCNA-unloading domain (ULD) of ATAD5. We identified several motifs in the ATAD5 ULD that are essential in the PCNA-unloading process. The C-terminus of ULD is required for the stable association of RFC2-5 for active RLC formation. The N-terminus of ULD participates in the opening of the PCNA ring. ATAD5-RLC was more robustly bound to open-liable PCNA compared to the wild type. These results suggest that distinct motifs of the ATAD5 ULD participate in each step of the PCNA-unloading process