Oleg N. Voloshin

Homologous DNA Pairing Promoted by a 20-Amino Acid Peptide Derived from RecA

The molecular structure of the Escherichia coli RecA protein in the absence of DNA revealed two disordered or mobile loops that were proposed to be DNA binding sites. A short peptide spanning one of these loops was shown to carry out the key reaction mediated by the whole RecA protein: pairing (targeting) of a single-stranded DNA to its homologous site on a duplex DNA. In the course of the reaction the peptide bound to both substrate DNAs, unstacked the single-stranded DNA, and assumed a β structure. These events probably recapitulate the underlying molecular pathway or mechanism used by homologous recombination proteins.

The DinG Protein from Escherichia coli Is a Structure-specific Helicase

The Escherichia coli DinG protein is a DNA damage-inducible member of the helicase superfamily 2. Using a panel of synthetic substrates, we have systematically investigated structural requirements for DNA unwinding by DinG. We have found that the helicase does not unwind blunt-ended DNAs or substrates with 3′-ss tails. On the other hand, the 5′-ss tails of 11-15 nucleotides are sufficient to initiate DNA duplex unwinding; bifurcated substrates further facilitate helicase activity. DinG is active on 5′-flap structures; however, it is unable to unwind 3′-flaps. Similarly to the homologous Saccharomyces cerevisiae Rad3 helicase, DinG unwinds DNA·RNA duplexes. DinG is active on synthetic D-loops and R-loops. The ability of the enzyme to unwind D-loops formed on superhelical plasmid DNA by the E. coli recombinase RecA suggests that D-loops may be natural substrates for DinG. Although the availability of 5′-ssDNA tails is a strict requirement for duplex unwinding by DinG, the unwinding of D-loops can be initiated on substrates without any ss tails. Since DinG is DNA damage-inducible and is active on D-loops and forked structures, which mimic intermediates of homologous recombination and replication, we conclude that this helicase may be involved in recombinational DNA repair and the resumption of replication after DNA damage.

FANCJ helicase uniquely senses oxidative base damage in either strand of duplex DNA and is stimulated by replication protein A to unwind the damaged DNA substrate in a strand-specific manner.

FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism.

The resistance of DMC1 D-loops to dissociation may account for the DMC1 requirement in meiosis

The ubiquitously expressed Rad51 recombinase and the meiosis-specific Dmc1 recombinase promote the formation of strand-invasion products (D-loops) between homologous molecules. Strand-invasion products are processed by either the double-strand break repair (DSBR) or synthesis-dependent strand annealing (SDSA) pathway. D-loops destined to be processed by SDSA need to dissociate, producing non-crossovers, and those destined for DSBR should resist dissociation to generate crossovers. The mechanism that channels recombination intermediates into different homologous-recombination pathways is unknown. Here we show that D-loops in a human DMC1-driven reaction are substantially more resistant to dissociation by branch-migration proteins such as RAD54 than those formed by RAD51. We propose that the intrinsic resistance to dissociation of DMC1 strand-invasion intermediates may account for why DMC1 is essential to ensure the proper segregation of chromosomes in meiosis.

HOP2-MND1 modulates RAD51 binding to nucleotides and DNA

The HOP2-MND1 heterodimer is required for progression of homologous recombination in eukaryotes. In vitro, HOP2-MND1 stimulates the DNA strand exchange activities of RAD51 and DMC1. We demonstrate that HOP2-MND1 induces changes in the conformation of RAD51 that profoundly alter the basic properties of RAD51. HOP2-MND1 enhances the interaction of RAD51 with nucleotide cofactors and modifies its DNA binding specificity in a manner that stimulates DNA strand exchange. It enables RAD51 DNA strand exchange in the absence of divalent metal ions required for ATP binding and offsets the effect of the K133A mutation that disrupts ATP binding. During nucleoprotein formation HOP2-MND1 helps to load RAD51 on ssDNA restricting its dsDNA-binding and during the homology search it promotes dsDNA binding removing the inhibitory effect of ssDNA. The magnitude of the changes induced in RAD51 defines HOP2-MND1 as a “molecular trigger” of RAD51 DNA strand exchange.

The dual role of HOP2 in mammalian meiotic homologous recombination

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.

The duplex DNA is very underwound in the three-stranded RecA protein-mediated synaptic complex

The RecA protein is a central player in bacterial homologous recombination. It promotes two key events: the search for homology between two DNA molecules and the subsequent formation of the synaptic complex composed of RecA and three DNA strands (two from one duplex and one single strand). In spite of numerous studies, the architecture of the synaptic complex is still far from clear.

The L2 loop peptide of RecA stiffens and restricts base motions of single-stranded DNA similar to the intact protein1

The L2 loop in the RecA protein is the catalytic center for DNA strand exchange. Here we investigate the DNA binding properties of the L2 loop peptide using optical spectroscopy with polarized light. Both fluorescence intensity and anisotropy of an etheno‐modified poly(dA) increase upon peptide binding, indicate that the base motions of single‐stranded DNA are restricted in the complex. In agreement with this conclusion, the peptide‐poly(dT) complex exhibits a significant linear dichroism signal. The peptide is also found to modify the structure of double‐stranded DNA, but does not denature it. It is inferred that strand separation may not be required for the formation of a joint molecule.

Erratum: The resistance of DMC1 D-loops to dissociation may account for the DMC1 requirement in meiosis (Nature Structural and Molecular Biology (2011) 18 (56-60))

David Martin, Cristina Pantoja, Ana Fernández Miñán, Christian Valdes-Quezada, Eduardo Moltó, Fuencisla Matesanz, Ozren Bogdanović, Elisa de la Calle-Mustienes, Orlando Domínguez, Leila Taher, Mayra Furlan-Magaril, Antonio Alcina, Susana Cañón, María Fedetz, María A Blasco, Paulo S Pereira, Ivan Ovcharenko, Félix Recillas-Targa, Lluís Montoliu, Miguel Manzanares, Roderic Guigó, Manuel Serrano, Fernando Casares, & José Luis Gómez-Skarmeta Nat. Struct. Mol. Biol. 18, 708–714 (2011); published online 22 May 2011; corrected after print 3 June 2011.