Background: HDHB has been implicated in chromosomal replication, but its role has not been determined. Results: Replication stress recruits HDHB to chromatin in a checkpoint-independent, RPA-dependent manner, and HDHB silencing reduces recovery from replication stress. Conclusion: HDHB functions in chromosomal replication to relieve replication stress. Significance: HDHB competition with checkpoint-signaling proteins for binding to RPA may modulate cellular responses to replication stress.

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DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8–10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique sub-domain not found in the archaeal and bacterial primases. Calorimetry experiments reveal Mn2+ but not Mg2+ significantly enhances the binding of nucleotide to primase, which correlates with in vitro higher catalytic efficiency. The structure of p48 with bound UTP and Mn2+ provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding altered nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions were not viable. Our results revealed two residues (S160 and H166) in direct contact with the nucleotide that were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.

DNA polymerase ␣-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.De novo DNA replication begins with RNA primer synthesis on a DNA template, followed by primer extension and processive DNA synthesis. In prokaryotes, a primosome couples activity of primase with parental DNA unwinding by a hexameric helicase and a single-stranded DNA (ssDNA) 5 -binding protein, largely through dynamic physical associations among the three proteins (1, 2). In eukaryotes, the core of the primosome is the DNA polymerase ␣-primase complex (pol-prim), which catalyzes both RNA primer synthesis and extension into RNA-DNA primers (3). Pol-prim initiates synthesis of both leading and lagging strands at eukaryotic replication origins and is required during elongation to initiate synthesis of each Okazaki fragment on the lagging strand. Pol-prim also plays a vital role in telomere maintenance and intra-S phase checkpoint activity. However, the mechanisms that regulate recruitment and activity of pol-prim in these various settings remain poorly understood.Pol-prim is a complex of four subunits. Its primase subunits (p48 and p58) initially synthesize an 8 -12-nucleotide RNA primer, which is then shifted internally to the active site of the associated p180 DNA polymerase subunit for extension into a 30 -35-nucleotide RNA-DNA primer (4, 5). The fourth subunit of the pol-prim complex, known as p68 or B-subunit, lacks enzyme activity but is essential for S phase entr...

Replication of simian virus 40 (SV40) DNA, a model for eukaryotic chromosomal replication, can be reconstituted in vitro using the viral helicase (large tumor antigen, or Tag) and purified human proteins. Tag interacts physically with two cellular proteins, replication protein A and DNA polymerase ␣-primase (pol-prim), constituting the viral primosome. Like the well characterized primosomes of phages T7 and T4, this trio of proteins coordinates parental DNA unwinding with primer synthesis to initiate the leading strand at the viral origin and each Okazaki fragment on the lagging strand template. We recently determined the structure of a previously unrecognized pol-prim domain (p68N) that docks on Tag, identified the p68N surface that contacts Tag, and demonstrated its vital role in primosome function. Here, we identify the p68N-docking site on Tag by using structure-guided mutagenesis of the Tag helicase surface. A charge reverse substitution in Tag disrupted both p68N-binding and primosome activity but did not affect docking with other pol-prim subunits. Unexpectedly, the substitution also disrupted Tag ATPase and helicase activity, suggesting a potential link between p68N docking and ATPase activity. To assess this possibility, we examined the primosome activity of Tag with a single residue substitution in the Walker B motif. Although this substitution abolished ATPase and helicase activity as expected, it did not reduce pol-prim docking on Tag or primosome activity on single-stranded DNA, indicating that Tag ATPase is dispensable for primosome activity in vitro.De novo DNA replication begins with RNA primer synthesis on single-stranded template DNA, followed by primer extension by a processive DNA polymerase. In prokaryotic replication, the activity of the primase is coordinated with unwinding of duplex DNA by a hexameric replicative helicase and a single-stranded DNA (ssDNA) 2 -binding protein, largely through dynamic physical interactions among the three proteins, which constitute a primosome (1-4). In eukaryotes, the DNA polymerase ␣-primase (pol-prim) complex catalyzes both RNA primer synthesis and extension, yielding RNA-DNA primers of 30 -35 nucleotides (5, 6). Unlike the single subunit prokaryotic primases, pol-prim is a stable heterotetramer composed of the primase heterodimer p48/p58, the catalytic DNA polymerase subunit p180, and a regulatory subunit (B or p68) (7). The eukaryotic replicative helicase complex, Cdc45/ Mcm2-7/GINS, and the ssDNA-binding protein, replication protein A (RPA), appear to coordinate primer synthesis by polprim with parental DNA unwinding, as in prokaryotes (8 -12). However, the nature of the eukaryotic primosome and its operation during chromosome replication, telomere maintenance, and checkpoint signaling at stalled replication forks remain elusive.Because pol-prim is essential for replication of simian virus 40 (SV40) DNA, we utilize this model system here to investigate the functional architecture of a eukaryotic primosome. SV40 DNA replication can be reconstituted in c...