Robert D. Ott

CELL CYCLE-DEPENDENT REGULATION OF HUMAN DNA POLYMERASE ALPHA -PRIMASE ACTIVITY BY PHOSPHORYLATION

ABSTRACT DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

Insights into hRPA32 C-terminal domain--mediated assembly of the simian virus 40 replisome.

Simian virus 40 (SV40) provides a model system for the study of eukaryotic DNA replication, in which the viral protein, large T antigen (Tag), marshals human proteins to replicate the viral minichromosome. SV40 replication requires interaction of Tag with the host single-stranded DNA-binding protein, replication protein A (hRPA). The C-terminal domain of the hRPA32 subunit (RPA32C) facilitates initiation of replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR revealed physical interaction between hRPA32C and the Tag origin DNA–binding domain, and a structural model of the complex was determined. Point mutations were then designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact hRPA impaired initiation of replication and primosome activity, implying that this interaction has a critical role in assembly and progression of the SV40 replisome.

Structural mechanism of RPA loading on DNA during activation of a simple pre‐replication complex

We report that during activation of the simian virus 40 (SV40) pre‐replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single‐stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA‐binding domain (OBD) with the RPA high‐affinity ssDNA‐binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag‐OBD binds to RPA70AB at a site distal from the ssDNA‐binding sites and that RPA70AB, Tag‐OBD, and an 8‐nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8‐mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.

Functional Interactions among the Subunits of Replication Factor C Potentiate and Modulate Its ATPase Activity

Replication factor C (RF-C), a complex of five subunits, and several subassemblies of RF-C, representing intermediates along the proposed protein assembly pathway (Podust, V. N., and Fanning, E. (1997) J. Biol. Chem. 272, 6303–6310), were expressed in insect cells using baculoviruses encoding individual subunits (p140, p40, p38, p37, and p36). Purified proteins were analyzed for ATPase activity to assess the role of individual subunits in ATP hydrolysis. His-tagged p40 contained low ATPase activity, but tagged p37 and p36 did not. Complexes of p40·p37·p36 bearing a His tag on any subunit displayed DNA-stimulated ATPase activity, in agreement with a recent report (Cai, J., Gibbs, E., Uhlmann, F., Philips, B., Yao, N., O’Donnell, M., and Hurwitz, J. (1997)J. Biol. Chem. 272, 18974–18981). In contrast, complex p38·p37·p36-his displayed no ATPase, suggesting that p40 is essential for ATPase activity. Although p38 was not required for ATPase activity, the activity of the p40-his·p38·p37·p36 complex was more salt-resistant than that of the p40-his·p37·p36 complex. The p140 subunit further increased the specific ATPase activity of RF-C complex by enhancing its stimulation by DNA. Taken together, the data indicate that all five RF-C subunits constitute ATPase activity, although the contributions of the individual subunits differ. Predicted ATP-binding domains of all five subunits were mutated to assess the importance of multiple ATP-binding sites of RF-C. In each case, the Lys of the conserved P-loop motif was replaced by Glu. The ATP-binding domain of p38 was found to be dispensable for the activity of the five-subunit RF-C in polymerase δ DNA synthesis. In contrast, mutation of the ATP-binding domains in other RF-C subunits impaired RF-C assembly, function, or both.

A dominant-negative mutant of human DNA helicase B blocks the onset of chromosomal DNA replication.

A cDNA encoding a human ortholog of mouse DNA helicase B, which may play a role in DNA replication, has been cloned and expressed as a recombinant protein. The predicted human DNA helicase B (HDHB) protein contains conserved helicase motifs (superfamily 1) that are strikingly similar to those of bacterial recD and T4 dda proteins. The HDHB gene is expressed at low levels in liver, spleen, kidney, and brain and at higher levels in testis and thymus. Purified recombinant HDHB hydrolyzed ATP and dATP in the presence of single-stranded DNA, displayed robust 5′-3′ DNA helicase activity, and interacted physically and functionally with DNA polymerase α-primase. HDHB proteins with mutations in the Walker A or B motif lacked ATPase and helicase activity but retained the ability to interact with DNA polymerase α-primase, suggesting that the mutants might be dominant over endogenous HDHB in human cells. When purified HDHB protein was microinjected into the nucleus of cells in early G1, the mutant proteins inhibited DNA synthesis, whereas the wild type protein had no effect. Injection of wild type or mutant protein into cells at G1/S did not prevent DNA synthesis. The results suggest that HDHB function is required for S phase entry.

Role of the p68 Subunit of Human DNA Polymerase α-Primase in Simian Virus 40 DNA Replication

ABSTRACT DNA polymerase α-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.

The trypanosome alternative oxidase exists as a monomer in Trypanosoma brucei mitochondria

The bloodstream forms of African trypanosomes solely depend on trypanosome alternative oxidase (TAO), for respiration. Similar to alternative oxidases (AOXs) found in plants and fungi, TAO is a membrane-bound diiron protein. Here, we investigated if TAO exists as a dimer like plant AOXs, or as a monomer like that of fungi. We have found that TAO forms a homo-dimer on a regular SDS-PAGE in the absence of any reducing agent and exists as a monomer under reducing condition. However, TAO does not form a dimer upon treatment of mitochondria with diamide. TAO was found as a higher molecular mass complex on a Blue-native gel after solubilization with digitonin. In the detergent soluble form, TAO activity was stimulated under reducing and inhibited under oxidizing condition. However, these conditions have no effect on the TAO activity in the mitochondria. Moreover, chemical cross-linking analysis revealed that TAO could not be cross-linked when present in the mitochondria. Together, it suggests that like certain other hydrophobic membrane proteins, TAO forms a dimer or oligomer when solubilized with detergents, and the TAO-dimer is SDS-resistant. However, it exists as a monomer in Trypanosoma brucei mitochondria.

Mutational Analysis of Simian Virus 40 T-Antigen Primosome Activities in Viral DNA Replication

ABSTRACT The recruitment of DNA polymerase α-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.