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 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 Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

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 Topoisomerase III Acts at the Replication Fork To Remove Precatenanes

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Chong M. Lee ◽  
Guanshi Wang ◽  
Alexandros Pertsinidis ◽  
Kenneth J. Marians
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Dna Helicase ◽  
Holliday Junctions ◽  
Bacterial Cells ◽  
Topoisomerase Iv ◽  
Content Type ◽  
E Coli ◽  
Topoisomerase Iii

ABSTRACTThe role of DNA topoisomerase III (Topo III) in bacterial cells has proven elusive. Whereas eukaryotic Top IIIα homologs are clearly involved with homologs of the bacterial DNA helicase RecQ in unraveling double Holliday junctions, preventing crossover exchange of genetic information at unscheduled recombination intermediates, and Top IIIβ homologs have been shown to be involved in regulation of various mRNAs involved in neuronal function, there is little evidence for similar reactions in bacteria. Instead, most data point to Topo III playing a role supplemental to that of topoisomerase IV in unlinking daughter chromosomes during DNA replication. In support of this model, we show thatEscherichia coliTopo III associates with the replication forkin vivo(likely via interactions with the single-stranded DNA-binding protein and the β clamp-loading DnaX complex of the DNA polymerase III holoenzyme), that the DnaX complex stimulates the ability of Topo III to unlink both catenated and precatenated DNA rings, and that ΔtopBcells show delayed and disorganized nucleoid segregation compared to that of wild-type cells. These data argue that Topo III normally assists topoisomerase IV in chromosome decatenation by removing excess positive topological linkages at or near the replication fork as they are converted into precatenanes.IMPORTANCETopological entanglement between daughter chromosomes has to be reduced to exactly zero every time anE. colicell divides. The enzymatic agents that accomplish this task are the topoisomerases.E. colipossesses four topoisomerases. It has been thought that topoisomerase IV is primarily responsible for unlinking the daughter chromosomes during DNA replication. We show here that topoisomerase III also plays a role in this process and is specifically localized to the replisome, the multiprotein machine that duplicates the cell’s genome, in order to do so.

scholarly journals Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Shun-Hsiao Lee ◽  
Lissa Nicola Princz ◽  
Maren Felizitas Klügel ◽  
Bianca Habermann ◽  
Boris Pfander ◽  
...  
Keyword(s):  
Dna Recognition ◽  
Dna Interaction ◽  
Holliday Junctions ◽  
Genome Integrity ◽  
Chromatin Remodelers ◽  
Activity Structure ◽  
Dna Strands ◽  
Holliday Junction Resolvase

Holliday junctions (HJs) are key DNA intermediates in homologous recombination. They link homologous DNA strands and have to be faithfully removed for proper DNA segregation and genome integrity. Here, we present the crystal structure of human HJ resolvase GEN1 complexed with DNA at 3.0 Å resolution. The GEN1 core is similar to other Rad2/XPG nucleases. However, unlike other members of the superfamily, GEN1 contains a chromodomain as an additional DNA interaction site. Chromodomains are known for their chromatin-targeting function in chromatin remodelers and histone(de)acetylases but they have not previously been found in nucleases. The GEN1 chromodomain directly contacts DNA and its truncation severely hampers GEN1’s catalytic activity. Structure-guided mutations in vitro and in vivo in yeast validated our mechanistic findings. Our study provides the missing structure in the Rad2/XPG family and insights how a well-conserved nuclease core acquires versatility in recognizing diverse substrates for DNA repair and maintenance.

scholarly journals Targeted chromosomal Escherichia coli:dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness

2021 ◽  
Author(s):  
Megan S Behrmann ◽  
Himasha M Perera ◽  
Joy M Hoang ◽  
Trisha A Venkat ◽  
MICHAEL TRAKSELIS
Keyword(s):  
Conformational Changes ◽  
Genomic Stability ◽  
Dna Helicase ◽  
Exterior Surface ◽  
Genomic Stress ◽  
Dna Strands ◽  
Helicase Dnab ◽  
Enzyme Functions

Helicase regulation is vital for replisome progression, where the helicase enzyme functions to unwind duplex DNA and aids in the coordination of replication fork activities. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased DNA damage and chromosome complexity, less stable genomes, and ultimately less viable and fit strains. Notably, while two mutations stabilized fully constricted states, they have distinct effects on genomic stability, suggesting a complex relationship between helicase regulation mechanisms and faithful, efficient DNA replication. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving SEW and conformational changes and relates current mechanistic understanding to functional helicase behavior.

scholarly journals Targeted chromosomal Escherichia coli: dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness

PLoS Genetics ◽  
2021 ◽  
Vol 17 (11) ◽  
pp. e1009886
Author(s):  
Megan S. Behrmann ◽  
Himasha M. Perera ◽  
Joy M. Hoang ◽  
Trisha A. Venkat ◽  
Bryan J. Visser ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Replication Fork ◽  
Dna Helicase ◽  
Exterior Surface ◽  
Genomic Stress ◽  
Dna Strands ◽  
Helicase Dnab ◽  
Organismal Fitness

Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork.

scholarly journals PCNA activates the MutLγ endonuclease to promote meiotic crossing over

2020 ◽  
Author(s):  
Dhananjaya S. Kulkarni ◽  
Shannon Owens ◽  
Masayoshi Honda ◽  
Masaru Ito ◽  
Ye Yang ◽  
...  
Keyword(s):  
High Efficiency ◽  
Holliday Junctions ◽  
Crossing Over ◽  
Branch Points ◽  
Dna Mismatch ◽  
Central Tenet ◽  
Dna Strands ◽  
Double Holliday Junction

AbstractDuring meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation1. Defects in crossing over cause infertility, miscarriage and congenital disease. Accordingly, each pair of chromosomes attains at least one crossover through processes that designate and then implement crossing over with high efficiency2. At the DNA level, crossing over is implemented through the formation and biased resolution of double-Holliday Junction intermediates3–5. A central tenet of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands6. Although the endonuclease activity of the MutLγ complex has been implicated in crossover-biased resolution7–12, the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp, PCNA, is important for crossover-biased resolution. In vitro assays with human enzymes show that hPCNA and its loader hRFC are sufficient to activate the hMutLγ endonuclease under physiological conditions. In this context, the hMutLγ endonuclease is further stimulated by a co-dependent activity of the pro-crossover factors hEXO1 and hMutSγ, the latter of which binds Holliday junctions13. hMutLγ also specifically binds a variety of branched DNAs, including Holliday junctions, but canonical resolvase activity is not observed implying that the endonuclease incises adjacent to junction branch points to effect resolution. In vivo, we show that budding yeast RFC facilitates MutLγ-dependent crossing over. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover-resolution and DNA mismatch repair14–16 and evoke a novel model for crossover-specific dHJ resolution during meiosis.

Review: Modulation of chromatin structure by poly(ADP-ribosyl)ation

1988 ◽  
Vol 66 (6) ◽  
pp. 626-635 ◽  
Author(s):  
G. de Murcia ◽  
A. Huletsky ◽  
G. G. Poirier
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Histone H1 ◽  
Histone H2b ◽  
Repair Enzymes ◽  
Core Histones ◽  
Partial Dissociation ◽  
Chromatin Superstructure

Poly(ADP–ribose) polymerase is a nuclear enzyme that is highly conserved in eucaryotes. Its activity is totally dependent on the presence of DNA containing single or double stranded breaks. We have shown that this activation results in a decondensation of chromatin superstructure in vitro, which is caused mainly by hyper(ADP-ribosyl)ation of histone H1. In core particles, the modification of histone H2B leads to a partial dissociation of DNA from core histones. The conformational change of native chromatin by poly(ADP-ribosyl)ation is reversible upon degradation of the histone H1-bound poly(ADP–ribose) by poly(ADP–ribose) glycohydrolase. We propose that cuts produced in vivo on DNA during DNA repair activate poly(ADP–ribose) polymerase, which then synthesizes poly(ADP–ribose) on histone H1, in particular, and contributes to the opening of the 25-nm chromatin fiber, resulting in the increased accessibility of DNA to excision repair enzymes. This mechanism is fast and reversible.

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