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

Arunkumar, A.I.; Klimovich, V. Ya.; Jiang, Xianzhen; Ott, R.; Mizoue, Laura S.; Fanning, Ellen; Chazin, Walter J.

doi:10.1038/nsmb916

Cited by 85 publications

(133 citation statements)

References 51 publications

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“…Structural studies have indicated that the C terminus of RPA2 makes direct contacts with similar motifs in the repair and recombination factors XPA, UNG2, and Rad52 (50). SV40 large T antigen also interacts with this domain but through a different motif (51). Strikingly, interactions between aRPA and these different protein partners vary considerably.…”

Section: Discussionmentioning

confidence: 99%

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

Kemp

Mason

Carreira

et al. 2010

Journal of Biological Chemistry

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Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells. Replication protein A (RPA)3 is the major single-stranded DNA-binding protein in human cells (1-3). It is composed of subunits of 70, 32, and 14 kDa (RPA1, the 70-kDa subunit of RPA; RPA2; and RPA3, the 14-kDa subunit of RPA, respectively) and was originally identified as an essential component for simian virus 40 (SV40) replication (1). RPA has since been shown to be essential for DNA replication, DNA repair, recombination, and coordination of the cellular response to DNA damage.In addition to the three canonical subunits of RPA (RPA1, RPA2, and RPA3), the human genome contains an additional subunit called RPA4 that is 63% similar to RPA2.4 RPA4 was originally identified in a screen for proteins that interact with RPA1 (5). RPA4 is an intronless gene on the X chromosome, and RPA4 homologs with complete coding sequences are only found in primates and horse. 4 Initial analysis indicated that at least some human tissues express RPA4 protein, though its role in these tissues was not determined (5). RPA4 protein can substitute for RPA2 in the RPA complex, forming an alternative RPA complex (aRPA) that has biochemical properties similar to canonical RPA (6). Surprisingly, whereas RPA is essential for DNA synthesis in the SV40 replication system, aRPA failed to substitute for RPA and acted in a dominant-negative fashion to inhibit DNA replication in the presence of canonical RPA (6). In addition, studies of RPA2-depleted HeLa cells expressing RPA4 demonstrated that aRPA is not able to support S-phase progr...

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Section: Discussionmentioning

confidence: 99%

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

Kemp

Mason

Carreira

et al. 2010

Journal of Biological Chemistry

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“…One of the most critical primase interactions is with ssDNA templates coated with replication protein A (RPA). RPA is a modular protein, and its ability to directly interact with many different proteins facilitates assembly and progression of DNA processing machinery (19,20). For the simian virus 40 (SV40) model system, interactions of pol-prim with RPA and the SV40 large T-antigen (Tag) helicase are required for efficient initiation of replication on the SV40 genome (21,22).…”

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confidence: 99%

Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase

Vaithiyalingam

Warren

Eichman

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-Å resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/ dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints from NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated ssDNA and regulation of primase activity.he replisome is a dynamic assembly of proteins that carries out eukaryotic DNA replication. The first step in the initiation of DNA replication on both the leading and lagging strand ssDNA is the synthesis of a short (8-10 nucleotide) RNA primer by a polymerase termed a DNA primase (1-3). DNA primases are unique because they are the sole polymerases capable of de novo synthesis on a ssDNA template. Although integral to the replisome, the activity of DNA primase is not limited to DNA replication; primase is also an essential component of the DNA damage response and plays a role in telomere maintenance (4, 5).Although all primases serve a common function, their architectures and modes of interaction with other protein factors vary among species (6, 7). The human DNA primase is part of the heterotetrameric polymerase α-primase (pol-prim) complex, which generates short (∼30 nucleotide) stretches of DNA primed with chimeric RNA-DNA hybrids that are handed off to the primary replicative DNA polymerases. The p48 and p58 subunits of human pol-prim form the DNA primase heterodimer. Although the p48 subunit contains the RNA polymerase catalytic active site, both subunits are required for synthesis of short RNA primers (8, 9). The p58 subunit is proposed to play various roles, including stabilizing the p48 subunit, recognizing the DNA template, and promoting initiation and elongation of primers (9, 10).Considerable knowledge about the molecular mechanism of DNA priming has been obtained from structural studies of archaeal and bacterial primases (11, 12). In contrast, the mechanism of DNA priming ...

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“…Understanding the timed formation and significance of protein-protein interactions during the different steps of the initiation reaction remains an active field of research [22,29]. The peptides with their marked specificities should be helpful in future studies to delineate more precisely the importance of mutual interactions between RPA, Tag and Pol in this process.…”

Section: Discussionmentioning

confidence: 99%

“…Interaction sites of different strengths have been mapped to DBD-A and N-terminal sequences of p70 and to a winged helix-loop-helix motif in the C-terminus of p32 [18,21,22].…”

Section: Introductionmentioning

confidence: 99%

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication

Taneja

Boche

Hartmann³

et al. 2007

FEBS Letters

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Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase a-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein-protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end.

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

Cited by 85 publications

References 51 publications

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication

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