scholarly journals An emerging picture of FANCJ’s role in G4 resolution to facilitate DNA replication

NAR Cancer ◽  
2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Robert M Brosh ◽  
Yuliang Wu
Keyword(s):  
Dna Replication ◽  
Double Helix ◽  
Dna Helicase ◽  
Nucleic Acid Binding ◽  
Dna Helicases ◽  
Recent Advances ◽  
G4 Dna ◽  
Cellular Dna

Abstract A well-accepted hallmark of cancer is genomic instability, which drives tumorigenesis. Therefore, understanding the molecular and cellular defects that destabilize chromosomal integrity is paramount to cancer diagnosis, treatment and cure. DNA repair and the replication stress response are overarching paradigms for maintenance of genomic stability, but the devil is in the details. ATP-dependent helicases serve to unwind DNA so it is replicated, transcribed, recombined and repaired efficiently through coordination with other nucleic acid binding and metabolizing proteins. Alternatively folded DNA structures deviating from the conventional anti-parallel double helix pose serious challenges to normal genomic transactions. Accumulating evidence suggests that G-quadruplex (G4) DNA is problematic for replication. Although there are multiple human DNA helicases that can resolve G4 in vitro, it is debated which helicases are truly important to resolve such structures in vivo. Recent advances have begun to elucidate the principal helicase actors, particularly in cellular DNA replication. FANCJ, a DNA helicase implicated in cancer and the chromosomal instability disorder Fanconi Anemia, takes center stage in G4 resolution to allow smooth DNA replication. We will discuss FANCJ’s role with its protein partner RPA to remove G4 obstacles during DNA synthesis, highlighting very recent advances and implications for cancer therapy.

scholarly journals Mechanistic insights into Lhr helicase function in DNA repair

2020 ◽  
Vol 477 (16) ◽  
pp. 2935-2947
Author(s):  
Ryan J. Buckley ◽  
Kevin Kramm ◽  
Christopher D. O. Cooper ◽  
Dina Grohmann ◽  
Edward L. Bolt
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Dna Helicase ◽  
Holliday Junctions ◽  
Single Molecule Fret ◽  
Repair Enzymes ◽  
Dna Strands

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

scholarly journals Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

2006 ◽  
Vol 27 (3) ◽  
pp. 1017-1026 ◽  
Author(s):  
Bryan E. Snow ◽  
Maria Mateyak ◽  
Jana Paderova ◽  
Andrew Wakeham ◽  
Caterina Iorio ◽  
...  
Keyword(s):  
Telomere Length ◽  
Chromosomal Rearrangements ◽  
Dna Helicase ◽  
Negative Regulator ◽  
Wild Type ◽  
Genetic Redundancy ◽  
Telomeric Dna ◽  
Dna Helicases

ABSTRACT Pif1 is a 5′-to-3′ DNA helicase critical to DNA replication and telomere length maintenance in the budding yeast Saccharomyces cerevisiae. ScPif1 is a negative regulator of telomeric repeat synthesis by telomerase, and recombinant ScPif1 promotes the dissociation of the telomerase RNA template from telomeric DNA in vitro. In order to dissect the role of mPif1 in mammals, we cloned and disrupted the mPif1 gene. In wild-type animals, mPif1 expression was detected only in embryonic and hematopoietic lineages. mPif1 − / − mice were viable at expected frequencies, displayed no visible abnormalities, and showed no reproducible alteration in telomere length in two different null backgrounds, even after several generations. Spectral karyotyping of mPif1 − / − fibroblasts and splenocytes revealed no significant change in chromosomal rearrangements. Furthermore, induction of apoptosis or DNA damage revealed no differences in cell viability compared to what was found for wild-type fibroblasts and splenocytes. Despite a novel association of mPif1 with telomerase, mPif1 did not affect the elongation activity of telomerase in vitro. Thus, in contrast to what occurs with ScPif1, murine telomere homeostasis or genetic stability does not depend on mPif1, perhaps due to fundamental differences in the regulation of telomerase and/or telomere length between mice and yeast or due to genetic redundancy with other DNA helicases.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

scholarly journals Changes in the topology of DNA replication intermediates: Important discrepancies between in vitro and in vivo

BioEssays ◽  
2021 ◽  
Vol 43 (5) ◽  
pp. 2000309
Author(s):  
Jorge B. Schvartzman ◽  
Víctor Martínez ◽  
Pablo Hernández ◽  
Dora B. Krimer ◽  
María‐José Fernández‐Nestosa
Keyword(s):  
Dna Replication ◽  
Replication Intermediates

scholarly journals The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

2021 ◽  
Author(s):  
Dipti Vinayak Vernekar ◽  
Giordano Reginato ◽  
Céline Adam ◽  
Lepakshi Ranjha ◽  
Florent Dingli ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Dna Helicase ◽  
Clamp Loader ◽  
Repair Synthesis ◽  
Dna Repair Synthesis ◽  
Gene Conversions

Abstract Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

scholarly journals Selective Targeting of Epigenetic Readers and Histone Deacetylases in Autoimmune and Inflammatory Diseases: Recent Advances and Future Perspectives

2021 ◽  
Vol 11 (5) ◽  
pp. 336
Author(s):  
Mohammed Ghiboub ◽  
Ahmed M. I. Elfiky ◽  
Menno P. J. de Winther ◽  
Nicola R. Harker ◽  
David F. Tough ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Inflammatory Diseases ◽  
Selective Inhibition ◽  
In Vivo Studies ◽  
Beneficial Effects ◽  
Domain Specific ◽  
Recent Advances ◽  
Autoimmune And Inflammatory Diseases

Histone deacetylases (HDACs) and bromodomain-containing proteins (BCPs) play a key role in chromatin remodeling. Based on their ability to regulate inducible gene expression in the context of inflammation and cancer, HDACs and BCPs have been the focus of drug discovery efforts, and numerous small-molecule inhibitors have been developed. However, dose-limiting toxicities of the first generation of inhibitors, which typically target multiple HDACs or BCPs, have limited translation to the clinic. Over the last decade, an increasing effort has been dedicated to designing class-, isoform-, or domain-specific HDAC or BCP inhibitors, as well as developing strategies for cell-specific targeted drug delivery. Selective inhibition of the epigenetic modulators is helping to elucidate the functions of individual epigenetic proteins and has the potential to yield better and safer therapeutic strategies. In accordance with this idea, several in vitro and in vivo studies have reported the ability of more selective HDAC/BCP inhibitors to recapitulate the beneficial effects of pan-inhibitors with less unwanted adverse events. In this review, we summarize the most recent advances with these strategies, discussing advantages and limitations of these approaches as well as some therapeutic perspectives, focusing on autoimmune and inflammatory diseases.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

scholarly journals UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

2001 ◽  
Vol 12 (5) ◽  
pp. 1199-1213 ◽  
Author(s):  
Gregory G. Oakley ◽  
Lisa I. Loberg ◽  
Jiaqin Yao ◽  
Mary A. Risinger ◽  
Remy L. Yunker ◽  
...  
Keyword(s):  
Dna Replication ◽  
Uv Radiation ◽  
Excision Repair ◽  
Genetic Disorder ◽  
Protein A ◽  
Dna Recombination ◽  
Delayed Response ◽  
Replication Intermediates

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

scholarly journals DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

2004 ◽  
Vol 279 (44) ◽  
pp. 45586-45593 ◽  
Author(s):  
David Shechter ◽  
Carol Y. Ying ◽  
Jean Gautier
Keyword(s):  
Dna Replication ◽  
Neutralizing Antibodies ◽  
Atp Hydrolysis ◽  
Initial Period ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Origin Firing ◽  
Replicative Helicase ◽  
Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

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.

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