scholarly journals Characterization of Streptococcus pneumoniae PriA helicase and its ATPase and unwinding activities in DNA replication restart

2020 ◽  
Vol 477 (19) ◽  
pp. 3911-3922
Author(s):  
Min-Guan Lin ◽  
Yi-Ching Li ◽  
Chwan-Deng Hsiao
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Gc Content ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Loop Area ◽  
Replication Restart ◽  
Key Factor

DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein — a 3′ to 5′ superfamily-2 DNA helicase — is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities of Streptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses. By comparing various DNA substrates, we observed that SpPriA is unable to unwind duplex DNA with high GC content. We constructed a deletion mutant protein (SpPriAdeloop) from which the loop area of the DNA-binding domain of PriA had been removed. Functional assays on SpPriAdeloop revealed that the loop area is important in endowing DNA-binding properties on the helicase. We also show that the presence of DnaD loader protein is important for enhancing SpPriA ATPase and DNA unwinding activities.

scholarly journals FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress

2013 ◽  
Vol 200 (2) ◽  
pp. 141-149 ◽  
Author(s):  
Yeon-Tae Jeong ◽  
Mario Rossi ◽  
Lukas Cermak ◽  
Anita Saraf ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genome Stability ◽  
Protein A ◽  
Replication Stress ◽  
Genotoxic Stress ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Physical Interaction ◽  
Helicase Activity ◽  
Dna Replication Stress

Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)–binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

Binding properties of replication protein A from human and yeast cells

1992 ◽  
Vol 12 (7) ◽  
pp. 3050-3059 ◽  
Author(s):  
C Kim ◽  
R O Snyder ◽  
M S Wold
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Simian Virus ◽  
Yeast Cells ◽  
Origin Of Replication ◽  
Binding Properties ◽  
Single Stranded Dna

Replication protein A (RP-A; also known as replication factor A and human SSB), is a single-stranded DNA-binding protein that is required for simian virus 40 DNA replication in vitro. RP-A isolated from both human and yeast cells is a very stable complex composed of 3 subunits (70, 32, and 14 kDa). We have analyzed the DNA-binding properties of both human and yeast RP-A in order to gain a better understanding of their role(s) in DNA replication. Human RP-A has high affinity for single-stranded DNA and low affinity for RNA and double-stranded DNA. The apparent affinity constant of RP-A for single-stranded DNA is in the range of 10(9) M-1. RP-A has a binding site size of approximately 30 nucleotides and does not bind cooperatively. The binding of RP-A to single-stranded DNA is partially sequence dependent. The affinity of human RP-A for pyrimidines is approximately 50-fold higher than its affinity for purines. The binding properties of yeast RP-A are similar to those of the human protein. Both yeast and human RP-A bind preferentially to the pyrimidine-rich strand of a homologous origin of replication: the ARS307 or the simian virus 40 origin of replication, respectively. This asymmetric binding suggests that RP-A could play a direct role in the process of initiation of DNA replication.

scholarly journals Replication Protein A-Directed Unloading of PCNA by the Ctf18 Cohesion Establishment Complex

2005 ◽  
Vol 25 (13) ◽  
pp. 5445-5455 ◽  
Author(s):  
Göran O. Bylund ◽  
Peter M. J. Burgers
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Sister Chromatid ◽  
Protein A ◽  
Replication Protein A ◽  
Dna Binding Protein ◽  
Sister Chromatid Cohesion ◽  
Replication Protein ◽  
Single Stranded Dna ◽  
Chromatid Cohesion

ABSTRACT The replication clamp PCNA is loaded around DNA by replication factor C (RFC) and functions in DNA replication and repair. Regulated unloading of PCNA during the progression and termination of DNA replication may require additional factors. Here we show that a Saccharomyces cerevisiae complex required for the establishment of sister chromatid cohesion functions as an efficient unloader of PCNA. Unloading requires ATP hydrolysis. This seven-subunit Ctf18-RFC complex consists of the four small subunits of RFC, together with Ctf18, Dcc1, and Ctf8. Ctf18-RFC was also a weak loader of PCNA onto naked template-primer DNA. However, when the single-stranded DNA template was coated by the yeast single-stranded DNA binding protein replication protein A (RPA) but not by a mutant form of RPA or a heterologous single-stranded DNA binding protein, both binding of Ctf18-RFC to substrate DNA and loading of PCNA were strongly inhibited, and unloading predominated. Neither yeast RFC itself nor two other related clamp loaders, containing either Rad24 or Elg1, catalyzed significant unloading of PCNA. The Dcc1 and Ctf8 subunits of Ctf18-RFC, while required for establishing sister chromatid cohesion in vivo, did not function specifically in PCNA unloading in vitro, thereby separating the functionality of the Ctf18-RFC complex into two distinct paths.

scholarly journals The Inhibitory Action of P56 on Select Functions of E1 Mediates Interferon's Effect on Human Papillomavirus DNA Replication

2010 ◽  
Vol 84 (24) ◽  
pp. 13036-13039 ◽  
Author(s):  
Paramananda Saikia ◽  
Volker Fensterl ◽  
Ganes C. Sen
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Dna Binding ◽  
Inhibitory Action ◽  
Dna Helicase ◽  
Single Amino Acid ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Hpv Dna ◽  
Papillomavirus Dna

ABSTRACT The interferon (IFN)-induced protein P56 inhibits human papillomavirus (HPV) DNA replication by binding to HPV E1, which has several distinct functions in initiating viral DNA replication. Here, we determined that P56 inhibited HPV type 18 (HPV18) E1's DNA helicase activity, E2 binding, and HPV Ori sequence-specific DNA binding but not nonspecific DNA binding. We observed that deletion of a single amino acid, F399, produced an E1 mutant that could not bind P56. This E1 mutant retained its ability to support Ori DNA replication, but this activity was not inhibited by IFN, demonstrating that P56 is the principal executor of the anti-HPV action of IFN.

scholarly journals Human HEL308 Localizes to Damaged Replication Forks and Unwinds Lagging Strand Structures

2011 ◽  
Vol 286 (18) ◽  
pp. 15832-15840 ◽  
Author(s):  
Agnieszka A. Tafel ◽  
Leonard Wu ◽  
Peter J. McHugh
Keyword(s):  
Protein A ◽  
Dna Helicase ◽  
Replication Forks ◽  
Double Stranded Dna ◽  
Replication Restart ◽  
Two Factors ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Stalled Replication Forks ◽  
Lagging Strand

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3′ to 5′ polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3′ single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

scholarly journals PriC-mediated DNA Replication Restart Requires PriC Complex Formation with the Single-stranded DNA-binding Protein

2013 ◽  
Vol 288 (24) ◽  
pp. 17569-17578 ◽  
Author(s):  
Sarah R. Wessel ◽  
Aimee H. Marceau ◽  
Shawn C. Massoni ◽  
Ruobo Zhou ◽  
Taekjip Ha ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
Replication Restart ◽  
Dna Replication Restart

scholarly journals Replication protein A binds RNA and promotes R-loop formation

2020 ◽  
Author(s):  
Olga M. Mazina ◽  
Srinivas Somarowthu ◽  
Lyudmila Y. Kadyrova ◽  
Andrey G. Baranovskiy ◽  
Tahir H. Tahirov ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Loop Formation ◽  
Genome Maintenance ◽  
Ssdna Binding ◽  
Replication Restart ◽  
Ssdna Binding Protein

SUMMARYReplication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA including DNA replication, repair, and damage signaling. Surprisingly, we found here that RPA binds RNA in vitro with high affinity. Using native RIP method, we isolated RNA-RPA complexes from human cells. Furthermore, RPA promotes R-loop formation between RNA and homologous dsDNA. R-loops, the three-stranded nucleic acid structure consisting of an RNA-DNA hybrid and the displaced ssDNA strand, are common in human genome. R-loops may play an important role in transcription-coupled homologous recombination and DNA replication restart. We reconstituted the process of replication restart in vitro using RPA-generated R-loops and human DNA polymerases. These findings indicate that RPA may play a role in RNA metabolism and suggest a mechanism of genome maintenance that depends on RPA and RNA.

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