scholarly journals The N-Terminal Noncatalytic Region of Xenopus RecQ4 Is Required for Chromatin Binding of DNA Polymerase α in the Initiation of DNA Replication

2006 ◽  
Vol 26 (13) ◽  
pp. 4843-4852 ◽  
Author(s):  
Kumiko Matsuno ◽  
Maya Kumano ◽  
Yumiko Kubota ◽  
Yoshitami Hashimoto ◽  
Haruhiko Takisawa
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Sequence Similarity ◽  
Dna Polymerases ◽  
Helicase Domain ◽  
Chromatin Binding ◽  
Replication Machinery ◽  
Dna Polymerase Α ◽  
Initiation Of Dna Replication ◽  
Replication Activity

ABSTRACT Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases α and ε onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase α. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.

scholarly journals Plant DNA polymerases alpha and delta mediate replication of geminiviruses

2020 ◽  
Author(s):  
Mengshi Wu ◽  
Hua Wei ◽  
Huang Tan ◽  
Shaojun Pan ◽  
Qi Liu ◽  
...  
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Dna Polymerase ◽  
Plant Cell ◽  
Dna Polymerases ◽  
Infected Plant ◽  
Replication Machinery ◽  
Dna Polymerase Δ ◽  
Dna Polymerase Α ◽  
Productive Replication

ABSTRACTGeminiviruses are causal agents of devastating diseases in crops. Geminiviruses have circular single-stranded (ss)DNA genomes that are replicated in the nucleus of the infected plant cell through double-stranded (ds)DNA intermediates by the plant’s DNA replication machinery; which host DNA polymerase mediates geminiviral multiplication, however, has so far remained elusive. Here, we show that subunits of the nuclear replicative DNA polymerases α and δ physically interact with the geminivirus-encoded replication enhancer protein, C3, and are required for viral replication. Our results suggest that while DNA polymerase α is essential to generate the viral dsDNA intermediate, DNA polymerase δ mediates the synthesis of new copies of the geminiviral ssDNA genome, and that the virus-encoded C3 acts selectively recruiting DNA polymerase δ over ε to favour a productive replication.

scholarly journals GINS Inactivation Phenotypes Reveal Two Pathways for Chromatin Association of Replicative α and ε DNA Polymerases in Fission Yeast

2009 ◽  
Vol 20 (4) ◽  
pp. 1213-1222 ◽  
Author(s):  
Chen Chun Pai ◽  
Ignacio García ◽  
Shao Win Wang ◽  
Sue Cotterill ◽  
Stuart A. MacNeill ◽  
...  
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Chromatin Binding ◽  
Dna Polymerase Α ◽  
Replication Arrest ◽  
Eukaryotic Dna ◽  
Separate Pathway ◽  
Hormone Binding Domain

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of β-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase ε, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase α is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase ε chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase α.

scholarly journals The B-Subunit of DNA Polymerase α-Primase Associates with the Origin Recognition Complex for Initiation of DNA Replication

2004 ◽  
Vol 24 (17) ◽  
pp. 7419-7434 ◽  
Author(s):  
Masashi Uchiyama ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Dna Polymerase ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Dna Polymerase Α ◽  
B Subunit ◽  
Initiation Of Dna Replication ◽  
Primase Complex ◽  
Origin Recognition

ABSTRACT The B-subunit (p70/Pol12p) of the DNA polymerase α-primase (Polα-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polα-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polα-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polα-primase in the initiation of both leading and lagging strands at the replication origins.

scholarly journals Plant DNA polymerases α and δ mediate replication of geminiviruses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mengshi Wu ◽  
Hua Wei ◽  
Huang Tan ◽  
Shaojun Pan ◽  
Qi Liu ◽  
...  
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Dna Polymerase ◽  
Plant Cell ◽  
Dna Polymerases ◽  
Infected Plant ◽  
Replication Machinery ◽  
Dna Polymerase Δ ◽  
Plant Dna ◽  
Productive Replication

AbstractGeminiviruses are causal agents of devastating diseases in crops. Geminiviruses have circular single-stranded (ss) DNA genomes that are replicated in the nucleus of the infected plant cell through double-stranded (ds) DNA intermediates by the plant DNA replication machinery. Which host DNA polymerase mediates geminiviral multiplication, however, has so far remained elusive. Here, we show that subunits of the nuclear replicative DNA polymerases α and δ physically interact with the geminivirus-encoded replication enhancer protein, C3, and that these polymerases are required for viral replication. Our results suggest that, while DNA polymerase α is essential to generate the viral dsDNA intermediate, DNA polymerase δ mediates the synthesis of new copies of the geminiviral ssDNA genome, and that the virus-encoded C3 may act selectively, recruiting DNA polymerase δ over ε to favour productive replication.

scholarly journals Multiple Phosphorylation Sites of DNA Polymerase α-Primase Cooperate to Regulate the Initiation of DNA Replication in Vitro

2001 ◽  
Vol 276 (41) ◽  
pp. 38076-38083
Author(s):  
Oliver Schub ◽  
Gabor Rohaly ◽  
Richard W.P. Smith ◽  
Annerose Schneider ◽  
Silke Dehde ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Phosphorylation Sites ◽  
Dna Polymerase Α ◽  
Initiation Of Dna Replication

scholarly journals Archaeal DNA Replication: Identifying the Pieces to Solve a Puzzle

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1249-1267 ◽  
Author(s):  
Isaac K O Cann ◽  
Yoshizumi Ishino
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Dna Polymerases ◽  
Small Subunit ◽  
Accessory Proteins ◽  
Pol Ii

Abstract Archaeal organisms are currently recognized as very exciting and useful experimental materials. A major challenge to molecular biologists studying the biology of Archaea is their DNA replication mechanism. Undoubtedly, a full understanding of DNA replication in Archaea requires the identification of all the proteins involved. In each of four completely sequenced genomes, only one DNA polymerase (Pol BI proposed in this review from family B enzyme) was reported. This observation suggested that either a single DNA polymerase performs the task of replicating the genome and repairing the mutations or these genomes contain other DNA polymerases that cannot be identified by amino acid sequence. Recently, a heterodimeric DNA polymerase (Pol II, or Pol D as proposed in this review) was discovered in the hyperthermophilic archaeon, Pyrococcus furiosus. The genes coding for DP1 and DP2, the subunits of this DNA polymerase, are highly conserved in the Euryarchaeota. Euryarchaeotic DP1, the small subunit of Pol II (Pol D), has sequence similarity with the small subunit of eukaryotic DNA polymerase δ. DP2 protein, the large subunit of Pol II (Pol D), seems to be a catalytic subunit. Despite possessing an excellent primer extension ability in vitro, Pol II (Pol D) may yet require accessory proteins to perform all of its functions in euryarchaeotic cells. This review summarizes our present knowledge about archaeal DNA polymerases and their relationship with those accessory proteins, which were predicted from the genome sequences.

scholarly journals A C-Terminal Helicase Domain of the Human Papillomavirus E1 Protein Binds E2 and the DNA Polymerase α-Primase p68 Subunit

1998 ◽  
Vol 72 (9) ◽  
pp. 7407-7419 ◽  
Author(s):  
Philip J. Masterson ◽  
Margaret A. Stanley ◽  
Alan P. Lewis ◽  
Michael A. Romanos
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Deletion Analysis ◽  
Nucleoside Triphosphate ◽  
Structural Domain ◽  
Helicase Domain ◽  
Sequence Alignments ◽  
Hpv 16 ◽  
Dna Polymerase Α

ABSTRACT The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase α-primase (polα-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polα-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind theori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polα-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

2020 ◽  
Vol 2 (4) ◽  
pp. 89-92
Author(s):  
Muhammad Amir ◽  
Sabeera Afzal ◽  
Alia Ishaq
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Information ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Polymerases ◽  
Test Site ◽  
Mitochondrial Dna Replication ◽  
Dna Template ◽  
Preliminary Phase

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol  DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

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