Role Of Replication Protein Research Articles

Role of Replication Protein A in Double Holliday Junction Dissolution Mediated by the BLM-Topo IIIα-RMI1-RMI2 Protein Complex

The conserved BTR complex, composed of the Bloom's syndrome helicase (BLM), topoisomerase IIIα, RMI1, and RMI2, regulates homologous recombination in favor of non-crossover formation via the dissolution of the double Holliday Junction (dHJ). Here we show enhancement of the BTR-mediated dHJ dissolution reaction by the heterotrimeric single-stranded DNA binding protein replication protein A (RPA). Our results suggest that RPA acts by sequestering a single-stranded DNA intermediate during dHJ dissolution. We provide evidence that RPA physically interacts with RMI1. The RPA interaction domain in RMI1 has been mapped, and RMI1 mutants impaired for RPA interaction have been generated. Examination of these mutants ascertains the significance of the RMI1-RPA interaction in dHJ dissolution. Our results thus implicate RPA as a cofactor of the BTR complex in dHJ dissolution.

Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts

Uncoupling between DNA polymerases and helicase activities at replication forks, induced by diverse DNA lesions or replication inhibitors, generate long stretches of primed single-stranded DNA that is implicated in activation of the S-phase checkpoint. It is currently unclear whether nucleation of the essential replication factor RPA onto this substrate stimulates the ATR-dependent checkpoint response independently of its role in DNA synthesis. Using Xenopus egg extracts to investigate the role of RPA recruitment at uncoupled forks in checkpoint activation we have surprisingly found that in conditions in which DNA synthesis occurs, RPA accumulation at forks stalled by either replication stress or UV irradiation is dispensable for Chk1 phosphorylation. In contrast, when both replication fork uncoupling and RPA hyperloading are suppressed, Chk1 phosphorylation is inhibited. Moreover, we show that extracts containing reduced levels of RPA accumulate ssDNA and induce spontaneous, caffeine-sensitive, Chk1 phosphorylation in S-phase. These results strongly suggest that disturbance of enzymatic activities of replication forks, rather than RPA hyperloading at stalled forks, is a critical determinant of ATR activation.

Binding activity of replication protein A to single-stranded DNA containing oxidized pyrimidine base.

To obtain the information for the role of replication protein A (RPA) on the detection of oxidized lesion in the single-stranded DNA, the binding preference of RPA purified from Xenopus egg lysate against the oligonucleotide containing one of three kinds of oxidized thymine residues, 5-formyluracil, 5-hydroxymethyluracil and 5-(1,2-dihydroxyethyl)uracil, was studied by the gel shift assay. Results of competition assay indicate that RPA preferentially binds to the oligonucleotide containing these oxidized thymine residues than the undamaged DNA.

Binding activity of replication protein A to UV-damaged single-stranded DNA.

To obtain the information for the exact role of replication protein A (RPA) on both eukaryotic DNA replication and repair, the binding preference of RPA purified from Xenopus egg extract against the undamaged and UV-damaged single-stranded DNA was studied by the gel shift assay. Chemically synthesized oligonucleotide containing the pyrimidine(6-4)pyrimidone photoproduct at one site was used as a model of UV-damaged DNA. Results of competition assay and Scatchard plots indicate that RPA preferentially binds to the 6-4 photoproduct oligonucleotide than the undamaged DNA.

The role of replication proteins in the regulation of bacteriophage T4 transcription: II. Gene 45 and late transcription uncoupled from replication

In this paper we have further analyzed the role of individual replication proteins in phage T4 late gene expression. One special objective has been to determine whether individual replication gene products affect late gene expression that has independently been uncoupled from replication. In the experiments which achieve this objective, T4 pol ts post− lig am post− exof am post− amX phage have been constructed, with mutations X in the various replication genes. Viral RNA and proteins have been analyzed by hybridization-competition and by acrylamide gel electrophoresis respectively. Mutations in the replication genes have been found to produce two kinds of effects: (1) a mutation in gene 45 almost completely abolishes late gene expression. Only the gene 45 mutation does this. Gene 45 protein is continuously required for late transcription. (2) Amber mutations in the other replication genes increase the temperature sensitivity of replication-independent late T4 transcription to varying degrees. The product of gene 45 is thus identified as a third essential component of late transcription, together with the previously identified gene 33 and 55 proteins. The endonucleolytic cleavage of parental DNA in the absence of phage DNA replication has also been examined. The distribution of single-strand breaks between the complementary DNA strands is not appreciably asymmetric. Breaks are introduced into the parental DNA of all phage mutants examined, in which no DNA replication occurs, including a mutant in gene 45. In the closing discussion we attempt to analyze the processing of T4 DNA for selective gene expression. The possibility that this processing and late transcription involve a complex of many replication proteins is examined.

The role of replication proteins in the regulation of bacteriophage T4 transcription: I. Gene 45 and hydroxymethyl-C-containing DNA

In this and the following paper, expression of the phage T4 late genes in the absence of DNA replication has been examined. With only one exception, mutants that block viral replication allow synthesis of late RNA and late proteins. The peculiar character of the DNA replication-independent T4 late transcription (measured, in our experiments, as RNA that hybridizes with the isolated r-strand of T4 DNA) is the dependence of the proportion of r-transcription on the number of infecting phage and the temperature sensitivity of this transcription. Mutants in gene 45 fail to synthesize appreciable proportions of late RNA. The effects of mutations in gene 45 on late transcription are more severe than those in the other hitherto identified T4 late transcription control genes, 33 or 55. We conclude (see also the following paper by Wu et al., 1975) that the product of T4 gene 45 is required for T4 late transcription as well as for DNA replication. Mutants in four T4 DNA genes—30, 41, 46 and 56—have defective replication but synthesize some viral DNA. Mutations in three of these genes, 30, 41 and 46, allow the synthesis of T4 late RNA at levels that do not depend on the multiplicity of infection. Mutations in gene 56 (which codes for a dCTPase) lead to the synthesis of T4 DNA containing C in place of hydroxymethyl. Such DNA is subject to degradation by virus-specified endo- and exonucleases. In cells infected with a gene 56 mutant, some r-strand-specific T4 RNA is synthesized but synthesis of late T4 proteins is defective. We provide evidence that this is an intrinsic property of C-containing T4 DNA rather than a consequence of its susceptibility to degradation by phage-specified nucleases.