Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.

Download Full-text

Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: Comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase ? activity

Journal of Cellular Physiology ◽

10.1002/jcp.1041340107 ◽

1988 ◽

Vol 134 (1) ◽

pp. 57-66 ◽

Cited By ~ 3

Author(s):

Angela M. Otto ◽

Christina Smith ◽

Luis Jimenez De Asua

Keyword(s):

Dna Replication ◽

Growth Factor ◽

Dna Synthesis ◽

Dna Polymerase ◽

Polymerase Activity ◽

S Phase ◽

3T3 Cells ◽

Swiss 3T3 ◽

Stimulation Of

Download Full-text

Two regions within the DNA binding domain of nuclear factor I interact with DNA and stimulate adenovirus DNA replication independently.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4073 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4073-4080 ◽

Cited By ~ 21

Author(s):

J Dekker ◽

J A van Oosterhout ◽

P C van der Vliet

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Dna Polymerase ◽

Binding Domain ◽

Nuclear Factor ◽

Dna Binding Domain ◽

Factor I ◽

Terminal Domain ◽

Nuclear Factor I ◽

Stimulation Of

The cellular transcription factor nuclear factor I (NFI) stimulates adenovirus DNA replication by up to 50-fold. The NFI DNA binding domain (NFI-BD) is sufficient for stimulation and interacts with the viral DNA polymerase, thereby recruiting the precursor terminal protein-DNA polymerase complex (pTP-pol) to the origin of replication. The mechanism of DNA binding by NFI is unknown. To examine DNA binding and stimulation of adenovirus DNA replication by NFI-BD in more detail, we generated a series of deletion mutants and show that the DNA binding domain of NFI consists of two subdomains: a highly basic N-terminal domain that binds nonspecifically to DNA and a C-terminal domain that binds specifically but with very low affinity to the NFI recognition site. Both of these subdomains stimulate DNA replication, although not to the same extent as the intact DNA binding domain. The N-terminal domain has an alpha-helical structure, as shown by circular dichroism spectroscopy. The C-terminal domain interacts with the pTP-pol complex and is able to recruit the pTP-pol complex to DNA, which leads to pTP-pol-dependent stimulation of replication. The N-terminal domain also stimulates replication in a pTP-pol-dependent manner and enhances binding of pTP-pol to DNA. Since we could not detect a direct protein-protein interaction between pTP-pol and the N-terminal domain, we suggest that this domain stimulates replication by inducing structural changes in the DNA.

Download Full-text

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Journal of Biological Chemistry ◽

10.1074/jbc.m109.050740 ◽

2009 ◽

Vol 284 (46) ◽

pp. 31672-31679 ◽

Cited By ~ 39

Author(s):

Quan Yuan ◽

Charles S. McHenry

Keyword(s):

Dna Binding ◽

Dna Polymerase ◽

Binding Protein ◽

Dna Binding Protein ◽

Dna Polymerase Iii ◽

Strand Displacement ◽

Single Stranded Dna ◽

Polymerase Iii ◽

Protein Coating ◽

Lagging Strand

Download Full-text

DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces cerevisiae

Genes ◽

10.3390/genes10020167 ◽

2019 ◽

Vol 10 (2) ◽

pp. 167 ◽

Cited By ~ 5

Author(s):

Michele Giannattasio ◽

Dana Branzei

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerases ◽

Experimental Results ◽

Genome Integrity ◽

Strand Displacement ◽

Viral Dna ◽

Okazaki Fragment Processing ◽

Lagging Strand

This review discusses a set of experimental results that support the existence of extended strand displacement events during budding yeast lagging strand DNA synthesis. Starting from introducing the mechanisms and factors involved in leading and lagging strand DNA synthesis and some aspects of the architecture of the eukaryotic replisome, we discuss studies on bacterial, bacteriophage and viral DNA polymerases with potent strand displacement activities. We describe proposed pathways of Okazaki fragment processing via short and long flaps, with a focus on experimental results obtained in Saccharomyces cerevisiae that suggest the existence of frequent and extended strand displacement events during eukaryotic lagging strand DNA synthesis, and comment on their implications for genome integrity.

Download Full-text

Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab205 ◽

2021 ◽

Author(s):

Natasha C Koussa ◽

Duncan J Smith

Keyword(s):

Transcription Factors ◽

Dna Polymerase ◽

Binding Sites ◽

Dna Ligase ◽

Strand Displacement ◽

Nick Translation ◽

Okazaki Fragment ◽

Dna Ligase I ◽

Lagging Strand

Abstract During lagging-strand synthesis, strand-displacement synthesis by DNA polymerase delta (Pol ∂), coupled to nucleolytic cleavage of DNA flap structures, produces a nick-translation reaction that replaces the DNA at the 5’ end of the preceding Okazaki fragment. Previous work following depletion of DNA ligase I in Saccharomyces cerevisae suggests that DNA-bound proteins, principally nucleosomes and the transcription factors Abf1/Rap1/Reb1, pose a barrier to Pol ∂ synthesis and thereby limit the extent of nick translation in vivo. However, the extended ligase depletion required for these experiments could lead to ongoing, non-physiological nick translation. Here, we investigate nick translation by analyzing Okazaki fragments purified after transient nuclear depletion of DNA ligase I in synchronized or asynchronous S. cerevisiae cultures. We observe that, even with a short ligase depletion, Okazaki fragment termini are enriched around nucleosomes and Abf1/Reb1/Rap1 binding sites. However protracted ligase depletion leads to a global change in the location of these termini, moving them towards nucleosome dyads from a more upstream location and further enriching termini at Abf1/Reb1/Rap1 binding sites. Additionally, we observe an under-representation of DNA derived from DNA polymerase alpha – the polymerase that initiates Okazaki fragment synthesis – around the sites of Okazaki termini obtained from very brief ligase depletion. Our data suggest that, while nucleosomes and transcription factors do limit strand-displacement synthesis by Pol ∂ in vivo, post-replicative nick translation can occur at unligated Okazaki fragment termini such that previous analyses represent an overestimate of the extent of nick translation occurring during normal lagging-strand synthesis.

Download Full-text

Pif1-family helicases cooperate to suppress widespread replication fork arrest at tRNA genes

10.1101/082008 ◽

2016 ◽

Author(s):

Joseph S. Osmundson ◽

Jayashree Kumar ◽

Rani Yeung ◽

Duncan J. Smith

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Polymerase ◽

Replication Fork ◽

Trna Genes ◽

Strand Displacement ◽

Dna Polymerase Δ ◽

Replication Fork Arrest ◽

Nuclear Processes ◽

Lagging Strand

ABSTRACTSaccharomyces cerevisiaeencodes two distinct Pif1-family helicases – Pif1 and Rrm3 – which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterize the roles of Pif1 helicases in replisome progression and lagging-strand synthesis inS. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulate strand-displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility inpif1andrrm3mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes; consistent with this observation, we find that head-on collisions between tRNA transcription and replisome progression are under-represented in theS. cerevisiaegenome. Further, we demonstrate that tRNA-mediated arrest is R-loop independent, and propose that replisome arrest and DNA damage are mechanistically separable.

Download Full-text

Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

10.1101/352567 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sarina Y. Porcella ◽

Natasha C. Koussa ◽

Colin P. Tang ◽

Daphne N. Kramer ◽

Priyanka Srivastava ◽

...

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Replication Fork ◽

Replication Initiation ◽

Replication Origins ◽

Origin Firing ◽

Leading Strand ◽

Strand Initiation ◽

Lagging Strand

AbstractDuring eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.Author summaryHalf of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

Download Full-text

High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1720353115 ◽

2018 ◽

Vol 115 (16) ◽

pp. 4212-4217 ◽

Cited By ~ 7

Author(s):

Katarzyna H. Maslowska ◽

Karolina Makiela-Dzbenska ◽

Jin-Yao Mo ◽

Iwona J. Fijalkowska ◽

Roel M. Schaaper

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Critical Factor ◽

Chromosomal Dna ◽

Experimental Systems ◽

Dna Strands ◽

Pol Iii ◽

Error Removal ◽

Lagging Strand

The fidelity of DNA replication is a critical factor in the rate at which cells incur mutations. Due to the antiparallel orientation of the two chromosomal DNA strands, one strand (leading strand) is replicated in a mostly processive manner, while the other (lagging strand) is synthesized in short sections called Okazaki fragments. A fundamental question that remains to be answered is whether the two strands are copied with the same intrinsic fidelity. In most experimental systems, this question is difficult to answer, as the replication complex contains a different DNA polymerase for each strand, such as, for example, DNA polymerases δ and ε in eukaryotes. Here we have investigated this question in the bacterium Escherichia coli, in which the replicase (DNA polymerase III holoenzyme) contains two copies of the same polymerase (Pol III, the dnaE gene product), and hence the two strands are copied by the same polymerase. Our in vivo mutagenesis data indicate that the two DNA strands are not copied with the same accuracy, and that, remarkably, the lagging strand has the highest fidelity. We postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by results with dnaE antimutator polymerases characterized by increased dissociation rates.

Download Full-text

Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

Journal of Virology ◽

10.1128/jvi.00581-12 ◽

2012 ◽

Vol 86 (18) ◽

pp. 9817-9827 ◽

Cited By ~ 19

Author(s):

Alexandra Nitzsche ◽

Charlotte Steinhäußer ◽

Katrin Mücke ◽

Christina Paulus ◽

Michael Nevels

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Histone Modification ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Viral Genome ◽

Histone H3 ◽

Viral Dna ◽

The Impact ◽

Preferential Incorporation

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using “click chemistry” revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.

Download Full-text