Deciphering the biochemical and biophysical properties of the Holliday junction resolvases RuvC and RuvX from Mycobacterium smegmatis

Abstract

Homologous recombination (HR) is a ubiquitous cellular process that occurs in all three domains of life as well as in DNA and RNA viruses. In eukaryotes, HR is critically important for homology-directed DNA repair (HDDR) in mitotic cells and synapsis of homologous chromosomes during prophase I of meiosis. Therefore, cells defective in HR exhibit multiple chromosomal aberrations, which raises the probability of genomic instability and cancer in eukaryotic organisms. In bacteria, similar to eukaryotes, HR is required for DNA repair and to facilitate horizontal gene transfer during transformation, transduction, and conjugation, while it plays a vital role in the evolution of viruses. A central intermediate formed during the late steps of HR and HDDR is a four-way joint DNA structure, also known as the Holliday junction (HJ), which is one among several different types of branched DNAs formed during HR and HDDR. A suite of studies have demonstrated that HJs adopt two global conformations: open and stacked. Whilst, the stacked form has two quasi-continuous helices and is induced by the binding of metal ions, the open form is a four-fold symmetric structure with a square, planar configuration, and is capable of branch migration between homologous duplexes, an essential step during DNA recombination and HDDR. The HJs are resolved by structure-specific endonucleases, known as junction resolvases, which are essential for faithful segregation of chromosomes during meiosis and double-strand break repair by HR in mitotic cells. Holliday junction resolvases (HJRs) have been identified in a wide variety of organisms, including animals, plants, fungi, and even microorganisms like bacteria and viruses based on their shared structural and functional characteristics, although they exhibit significant diversity and belong to different classes of structure-specific endonucleases. Genetic, functional and structural analyses of Escherichia coli RuvC, the founding member of canonical HJRs, has provided key insights into the mechanism of processing branched DNA intermediates that arise during HR and HDDR. In eukaryotes, three conserved nucleases, such as Mus81–Mms4/MUS81–EME1 (budding yeast/human) and Yen1/GEN1 and Slx1–Slx4/SLX1–SLX4 have been found to have properties similar to those of E. coli RuvC. In contrast to the large body of information on E. coli HJRs, our knowledge about the HJRs in Gram-positive bacteria, and in particular of mycobacterial species is incompletely understood. Of note, genetic studies have revealed that the mycobacterial HR pathway differs from that of HR in E. coli. Thus, a full understanding of the biophysical and biochemical characteristics of HJRs in mycobacterial species may reveal important insights into interspecies and intergenus divergence and evolution of their domain structure and function. A bioinformatics search on the whole genome sequence of Mycobacterium smegmatis revealed the presence of genes encoding two putative HJRs: ruvC and ruvX. Intuitively, this observation implies that ruvC and ruvX genes may play a crucial role in DNA recombination and repair. This finding raised intriguing questions: why does M. smegmatis encode teo putative HJRs; do they function independently or in a mutually exclusive manner? Thus, the overall purpose of our research is aimed at a fuller, more complete understanding of the functional aspects of M. smegmatis HJRs RuvC and RuvX. In the first half the thesis, we describe the molecular cloning, expression, purification and biochemical characterization of a canonical HJ resolvase RuvC from M. smegmatis (MsRuvC). The recombinant MsRuvC was purified to homogeneity using heparin affinity column and gel filtration chromatography under native conditions from the supernatant. The purified MsRuvC occurs as a homodimer in solution and shows binding preference to a wide variety of DNA structures, many of them mimic the intermediates generated either during recombination or replication. Importantly, we found that MsRuvC cleaves the mobile HJ (mHJ) at 5'-T↓C-3' around the crossover point, generating ligatable, nicked duplex products in Mg2+/Mn2+ dependent manner and resolution of three-way junctions occurred in the presence of Mn2+, but failed to act on immobile HJ, flaps, splayed arms, and replication fork structures. However, we cannot rule out the possibility that MsRuvC might cleave these substrates in the in vivo context. Our studies with various types of HJs revealed that MsRuvC-catalyzed incisions occur at the 3′ side of thymidine (5'-T↓C-3') positioned at or one base pair away from the branch point in HJ. We found that alanine substitution of Asp7 and Glu68 in the RNase-H domain of MsRuvC independently impaired the ability of MsRuvC to catalyse HJ resolution. However, their HJ binding activity remains unperturbed, indicating that binding by itself is not sufficient to ensure efficient HJ cleavage. Notably, MsRuvC was found to resolve the 3-way DNA junction as well, but failed to act on the Y-shaped substrate, flap and fork substrates, and duplex DNA. Overall, our data provide important insights into the substrate specificity of MsRuvC, mode of its substrate binding, as well as catalysis, advancing our knowledge on HJRs in mycobacteria. Viewed together, these findings indicate MsRuvC can be considered as a canonical HJ resolvase, similar to the prototype E. coli RuvC. Many studies have provided strong genetic evidence that certain bacterial and eukaryotic organisms encode enzymes belonging to the RNaseH/retroviral integrase superfamily, known as YqgF/RuvX proteins. Since YqgF shares striking sequence and structural similarities with RuvC, including the presence of an RNase H-like motif, it was predicted to function as an alternative HJR. Herein, we report the cloning of M. smegmatis ruvX, heterologous expression, and biophysical and biochemical characterization of recombinant MsRuvX, and compare the results with M. tuberculosis RuvX (MtRuvX). Unlike dimeric MtRuvX, MsRuvX exists as a monomer in solution and exhibits high binding affinity for a suite of branched DNAs. Rather unexpectedly, we found that MsRuvX has no HJ resolution activity, while delivering a very robust cleavage activity on a suite of branched DNAs, generating DNA products of different lengths in reaction buffers containing either Mg2+ or Ca2+. However, MsRuvX mediated flap resolution was found to be ~2 fold higher in presence of Mg2+ compared to that of Ca2+. Furthermore, MsRuvX promotes robust cleavage of single-stranded DNA (ssDNA), but not double-stranded DNA, and that it resects ssDNA in a 5'-to-3' direction. Mutations of amino acid residues D25 and D142 in the MsRuvX RNase H-like domain rendered the resulting variants functionally inactive; however, their DNA binding activity was unaffected. These results suggest that MsRuvX might play an important role in the processing of multiple types of branched DNAs, other than HJs, arising from cellular processes such as produced during HR and HDDR, thereby furthering our understanding of HR and HDDR in M. smegmatis. Overall, this study provides novel insights into the non-overlapping functions of RuvC and RuvX in the processing of a wide range of branched DNA structures in M. smegmatis. Importantly, this study has uncovered a strong division of labour between RuvC and RuvX in M. smegmatis, which may enhance the processing efficiency of different types of branched DNA structures. Thus, on the one hand, RuvC function might be limited to the resolution of HJ, on the other hand, RuvX may act in concert with, or independently from that of, RuvC in the processing of other types of branched DNAs that arise during the processes of HR and HDDR to maintain the integrity of M. smegmatis genome under different growth conditions.