Modeling the Homologous Recombination Process: Methods, Successes and Challenges

Abstract

Homologous recombination (HR) is a fundamental process common to all species. HR aims to faithfully repair DNA double strand breaks. HR involves the formation of nucleoprotein filaments on DNA single strands (ssDNA) resected from the break. The nucleoprotein filaments search for homologous regions in the genome and promote strand exchange with the ssDNA homologous region in an unbroken copy of the genome. HR has been the object of intensive studies for decades. Because multi-scale dynamics is a fundamental aspect of this process, studying HR is highly challenging, both experimentally and using computational approaches. Nevertheless, knowledge has built up over the years and has recently progressed at an accelerated pace, borne by increasingly focused investigations using new techniques such as single molecule approaches. Linking this knowledge to the atomic structure of the nucleoprotein filament systems and the succession of unstable, transient intermediate steps that takes place during the HR process remains a challenge; modeling retains a very strong role in bridging the gap between structures that are stable enough to be observed and in exploring transition paths between these structures. However, working on ever-changing long filament systems submitted to kinetic processes is full of pitfalls. This review presents the modeling tools that are used in such studies, their possibilities and limitations, and reviews the advances in the knowledge of the HR process that have been obtained through modeling. Notably, we will emphasize how cooperative behavior in the HR nucleoprotein filament enables modeling to produce reliable information.

Keywords: DNA stretching; RecA; homologous recombination; integrative modeling; molecular dynamics simulations; molecular modeling; multi-scale dynamics; protein filament; protein-DNA interaction.

Figure 1

Schematic representation of the components of the homologous recombination process at different length scale levels—protein, DNA, filament, DNA-filament complex and the modeling methods that are adapted to their study. The VMD software version 1.9.4a57 [23] was used for graphical representations in all Figures in this paper.

Figure 2

Modeling stretched DNA. (A) top, from left to right: B-DNA, S-form resulting from 3${}^{\prime }$-3${}^{\prime }$ pulling; S-form resulting from 5${}^{\prime }$-5${}^{\prime }$ pulling; melted DNA; bottom: schemes indicate the direction (arrows) and region (red lines) of application of the pulling force associated to the corresponding structure. (B) Binding to the TBP protein (orange, right panel) locally stretches the TATA-box DNA (red lines in the left panel scheme) generating kinks at the junction between relaxed (grey boxes) and locally stretched (red box) DNA regions (from crystal structure 1YTB). The DNA strands in (A) and (B) are colored blue and yellow. (C) Positions of the DNA strands in the synaptic (left) and post-synaptic (right) recombination filaments, after [87]; the outgoing, complementary and ssDNA strands are represented in cyan, blue and orange, respectively. The outgoing strand is not represented in the post-synaptic structure. The position of the outgoing and complementary strands in the synaptic structure correspond to axis separation values of 15 and 10 Å, respectively, as proposed by Egelman and Yu in 1989 (Figure 4 of [52], see also [88]). The positions of the heteroduplex strands in the post-synaptic structure, separated by 6 Å from the axis, were also predicted in that study.

Figure 3

RecA protein within the recombination filament. (A) Dynamic behavior of a RecA protein embedded in a RecA-ATP filament during a 200 ns molecular dynamic simulation, represented by the superposition of snapshots taken every 25 ns; regions that show higher flexibility (see typical RMSF plot in insert limited to the 1–333 region, adapted from [50]) are color-coded according to the labels and the dotted lines in the insert. (B) RecA-RecA interface change upon ATP hydrolysis. top panel: two filament forms observed in the presence of non-hydrolyzable ATP analogs (red, left), ADP or no cofactor (blue, right); monomers are colored alternatively with light and dark shades; bottom panel: two interacting monomers are represented in each view, with the bottom monomer in cartoon (N-terminal domain in purple, C-terminal domain in pink, ATPase domain in white) and the upper monomer in surface representation. The binding geometry in the left panel corresponds to the ATP-bound geometry (ATP is embedded in the interface) while the right panel shows the binding geometry corresponding to ADP as a cofactor or no cofactor.

Figure 4

RecA filament and strand exchange. (A) Model of the synaptic filament, adapted from [87]; the regions corresponding to the threshold regions, the 5${}^{\prime }$-and 3${}^{\prime }$ kink and the incorporated dsDNA in site II are lined with green, red, orange and black boxes, respectively. The protein monomers are not represented except for DNA-binding residues. the dsDNA strands in the homologous region of (A) and (B) are represented in cyan and blue for the outgoing and complementary strands, respectively, while the ssDNA is in orange. (B) Cryo-EM Structure of the post-synaptic filament, PDB ID 7JY9 [90]; RecA monomers are shown in cartoon representation and are alternatively colored lime and white; in the dsDNA heterologous bubble region, the outgoing strand is colored in black and the complementary strand backbone is in light blue; the complementary strand bases in that region form Watson-Crick interactions with the corresponding ssDNA bases in orange.

Figure 5

Electrostatic interactions created by RecA-ssDNA mini-filaments. (top) two views, rotated by 180°, of the electrostatic potential in the vicinity of the ssDNA-RecA Cryo-EM mini-filaments PDB ID 7JY9 in the absence of the dsDNA strands: surfaces corresponding to isopotential values of −0.8 and +0.8 are colored in red and blue, respectively. Strong electropositive potential observed at both ends of the mini-filament may favor DNA binding in those regions. (bottom) same view with the bound dsDNA shown in tube (backbone and licorice bases, with the outgoing strand in blue and the complementary strand in green). The potential was calculated using the APBS web server (https://server.poissonboltzmann.org, accessed on 10 August 2023) [126] and was represented with VMD [23].