scholarly journals Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination

2020 ◽  
Vol 117 (34) ◽  
pp. 20549-20554
Author(s):  
Xingyuan Huang ◽  
Ying Lu ◽  
Shuang Wang ◽  
Mingyu Sui ◽  
Jinghua Li ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Traveling Wave ◽  
Strand Exchange ◽  
Basic Unit ◽  
Base Pairs ◽  
Single Stranded Dna ◽  
Homologous Sequences ◽  
Transient Intermediates

Recombinases polymerize along single-stranded DNA (ssDNA) at the end of a broken DNA to form a helical nucleofilament with a periodicity of ∼18 bases. The filament catalyzes the search and checking for homologous sequences and promotes strand exchange with a donor duplex during homologous recombination (HR), the mechanism of which has remained mysterious since its discovery. Here, by inserting mismatched segments into donor duplexes and using single-molecule techniques to catch transient intermediates in HR, we found that, even though 3 base pairs (bp) is still the basic unit, both the homology checking and the strand exchange may proceed in multiple steps at a time, resulting in ∼9-bp large steps on average. More interestingly, the strand exchange is blocked remotely by the mismatched segment, terminating at positions ∼9 bp before the match–mismatch joint. The homology checking and the strand exchange are thus separated in space, with the strand exchange lagging behind. Our data suggest that the strand exchange progresses like a traveling wave in which the donor DNA is incorporated successively into the ssDNA–RecA filament to check homology in ∼9-bp steps in the frontier, followed by a hypothetical transitional segment and then the post-strand-exchanged duplex.

scholarly journals Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates

2019 ◽  
Vol 47 (21) ◽  
pp. 11225-11237 ◽  
Author(s):  
Chaoyou Xue ◽  
James M Daley ◽  
Xiaoyu Xue ◽  
Justin Steinfeld ◽  
Youngho Kwon ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Protein A ◽  
Base Pairs ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Blm Helicase ◽  
Dna Replication And Repair ◽  
Regulatory Effects

Abstract Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.

scholarly journals The Herpes Simplex Virus Type 1 Alkaline Nuclease and Single-Stranded DNA Binding Protein Mediate Strand Exchange In Vitro

2003 ◽  
Vol 77 (13) ◽  
pp. 7425-7433 ◽  
Author(s):  
Nina Bacher Reuven ◽  
Amy E. Staire ◽  
Richard S. Myers ◽  
Sandra K. Weller
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Homologous Recombination ◽  
Dna Binding Protein ◽  
Strand Exchange ◽  
Single Stranded Dna ◽  
Simplex Virus ◽  
Alkaline Nuclease ◽  
Hsv 1

ABSTRACT The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 5′→3′ exonuclease that shares homology with Redα, commonly known as λ exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redβ synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl2, 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.

scholarly journals Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA

2020 ◽  
Vol 21 (19) ◽  
pp. 7389
Author(s):  
Aleksandr Alekseev ◽  
Galina Cherevatenko ◽  
Maksim Serdakov ◽  
Georgii Pobegalov ◽  
Alexander Yakimov ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Deinococcus Radiodurans ◽  
Atp Hydrolysis ◽  
Conformational Dynamics ◽  
Strand Exchange ◽  
Single Stranded Dna ◽  
Dna Strand Exchange ◽  
High Conservation ◽  
Dna Strand

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

Single-molecule views on homologous recombination

2013 ◽  
Vol 46 (4) ◽  
pp. 323-348 ◽  
Author(s):  
Andrea Candelli ◽  
Mauro Modesti ◽  
Erwin J. G. Peterman ◽  
Gijs J. L. Wuite
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Genetic Instability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Strand Breaks ◽  
Future Developments ◽  
Recent Developments ◽  
Human Rad51

AbstractAll organisms need homologous recombination (HR) to repair DNA double-strand breaks. Defects in recombination are linked to genetic instability and to elevated risks in developing cancers. The central catalyst of HR is a nucleoprotein filament, consisting of recombinase proteins (human RAD51 or bacterial RecA) bound around single-stranded DNA. Over the last two decades, single-molecule techniques have provided substantial new insights into the dynamics of homologous recombination. Here, we survey important recent developments in this field of research and provide an outlook on future developments.

scholarly journals Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

2021 ◽  
Author(s):  
Sujay Ray ◽  
Nibedita Pal ◽  
Nils G Walter
Keyword(s):  
Cluster Analysis ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Holliday Junction ◽  
Energetic Cost ◽  
Genomic Integrity ◽  
Dna Molecules ◽  
Single Molecule Fluorescence ◽  
And Cluster Analysis ◽  
Fluorescence Observation

Abstract Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

scholarly journals The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Genetics ◽  
1989 ◽  
Vol 122 (4) ◽  
pp. 727-736
Author(s):  
C E Bauer ◽  
J F Gardner ◽  
R I Gumport ◽  
R A Weisberg
Keyword(s):  
Attachment Site ◽  
Strand Exchange ◽  
Segregation Pattern ◽  
Base Substitution ◽  
Base Pairs ◽  
Multiple Mutation ◽  
Attachment Sites ◽  
Dna Strands ◽  
Substitution Mutations ◽  
Overlap Region

Abstract Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

Effects of mutation position on frequency of marker rescue by homologous recombination

1992 ◽  
Vol 12 (8) ◽  
pp. 3609-3613
Author(s):  
L Jiang ◽  
A Connor ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Normal Function ◽  
Constant Region ◽  
Dna Fragments ◽  
Wild Type ◽  
End Effect ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Marker Rescue ◽  
Immunoglobulin Mu

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

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