scholarly journals A histone octamer blocks branch migration of a Holliday junction.

1997 ◽  
Vol 17 (12) ◽  
pp. 7139-7150 ◽  
Author(s):  
M Grigoriev ◽  
P Hsieh
Keyword(s):  
Genetic Recombination ◽  
Intrinsic Rate ◽  
Holliday Junctions ◽  
Micrococcal Nuclease ◽  
Holliday Junction ◽  
Sequence Motif ◽  
Branch Migration ◽  
Histone Octamers ◽  
Nuclease Mapping

The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.

Formation, translocation and resolution of Holliday junctions during homologous genetic recombination

1995 ◽  
Vol 347 (1319) ◽  
pp. 21-25 ◽  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Recombination ◽  
Holliday Junctions ◽  
The Novel ◽  
E Coli ◽  
The Past ◽  
Endonucleolytic Cleavage ◽  
Insight Into ◽  
Made In

Over the past three or four years, great strides have been made in our understanding of the proteins involved in recombination and the mechanisms by which recombinant molecules are formed. This review summarizes our current understanding of the process by focusing on recent studies of proteins involved in the later steps of recombination in bacteria. In particular, biochemical investigation of the in vitro properties of the E. coli RuvA, RuvB and RuvC proteins have provided our first insight into the novel molecular mechanisms by which Holliday junctions are moved along DNA and then resolved by endonucleolytic cleavage.

Structural analysis of the E. coli RUVB branch migration protein by EM and image analysis

1994 ◽  
Vol 52 ◽  
pp. 336-337
Author(s):  
X. Yu ◽  
K. Benson ◽  
A. Stasiak ◽  
I. Tsaneva ◽  
S. West ◽  
...  
Keyword(s):  
Image Analysis ◽  
Atp Hydrolysis ◽  
Genetic Recombination ◽  
Sos Response ◽  
Structure And Function ◽  
Holliday Junctions ◽  
Branch Migration ◽  
E Coli ◽  
Form Part ◽  
And Function

We have been interested in the structure and function of proteins involved in genetic recombinaton. The ruv locus on the E. coli chromosome contains three genes (ruvA, ruvB and ruvC) that are important for genetic recombination and DNA repair. The ruvA and ruvB genes form part of the SOS response to DNA damage and encode the RuvA and RuvB proteins. Together, RuvA and RuvB promote the branch migration of Holliday junctions in a reaction that requires ATP hydrolysis. Each protein plays a defined role, with RuvA responsible for DNA binding (and, in particular, junction recognition), whereas the RuvB ATPase provides the motor for branch migration. Sequence analysis has identified RuvB as a member of a superfamily of helicases, and experimentally it has been shown that RuvB, in the presence of RuvA, acts as an ATP-dependent helicase.When purified RuvB protein was incubated (in the presence of the ATP analog, ATP-γ-S) with covalently closed, relaxed dsDNA, double-ringed structures were observed on the DNA in the electron microscope (Fig. 1). The DNA must be passing through the center of these rings, since the rings are always aligned along a common axis.

scholarly journals Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 5099
Author(s):  
Saminathan Ramakrishnan ◽  
Sivaraman Subramaniam ◽  
Charlotte Kielar ◽  
Guido Grundmeier ◽  
A. Francis Stewart ◽  
...  
Keyword(s):  
Room Temperature ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Dna Nanotechnology ◽  
Holliday Junctions ◽  
Dna Nanostructures ◽  
Fundamental Building Block ◽  
In Vitro Investigation ◽  
Functional Dna

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA–protein nanostructures may find applications in the crystallization of DNA–protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

scholarly journals Polarity and Bypass of DNA Heterology during Branch Migration of Holliday Junctions by Human RAD54, BLM, and RECQ1 Proteins

2012 ◽  
Vol 287 (15) ◽  
pp. 11820-11832 ◽  
Author(s):  
Olga M. Mazina ◽  
Matthew J. Rossi ◽  
Julianna S. Deakyne ◽  
Fei Huang ◽  
Alexander V. Mazin
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Holliday Junctions ◽  
Holliday Junction ◽  
Helicase Activity ◽  
Branch Migration ◽  
Blm Helicase ◽  
Human Rad51

Several proteins have been shown to catalyze branch migration (BM) of the Holliday junction, a key intermediate in DNA repair and recombination. Here, using joint molecules made by human RAD51 or Escherichia coli RecA, we find that the polarity of the displaced ssDNA strand of the joint molecules defines the polarity of BM of RAD54, BLM, RECQ1, and RuvAB. Our results demonstrate that RAD54, BLM, and RECQ1 promote BM preferentially in the 3′→5′ direction, whereas RuvAB drives it in the 5′→3′ direction relative to the displaced ssDNA strand. Our data indicate that the helicase activity of BM proteins does not play a role in the heterology bypass. Thus, RAD54 that lacks helicase activity is more efficient in DNA heterology bypass than BLM or REQ1 helicases. Furthermore, we demonstrate that the BLM helicase and BM activities require different protein stoichiometries, indicating that different complexes, monomers and multimers, respectively, are responsible for these two activities. These results define BM as a mechanistically distinct activity of DNA translocating proteins, which may serve an important function in DNA repair and recombination.

Sign in / Sign up

Export Citation Format

Share Document