Deciphering the essentiality and function of SxSx motif in Mycobacterium tuberculosis UvrB

Highlights

• The UvrB SxSx motif is analogous to a TxGx motif in the helicases of superfamily 2.

• The function(s) of UvrB SxSx motif is unknown for orthologs across all species.

• To investigate the significance of SxSx motif, variants of UvrB were characterized.

• The SxSx motif of UvrB is involved in its DNA binding, helicase and ATPase activities.

Abstract

The UvrB subunit is a central component of the UvrABC incision complex and plays a pivotal role in damage recognition, strand excision and repair synthesis. A conserved structural motif (the SxSx motif) present in UvrB is analogous to a similar motif (TxGx) in the helicases of superfamily 2, whose function is not fully understood. To elucidate the significance of the SxSx (Ser143-Val144-Ser145-Cys146) motif in Mycobacterium tuberculosis UvrB (MtUvrB), different variants of MtUvrB subunit were constructed and characterized. The SxSx motif indeed was found to be essential for MtUvrB function: while Ser143 and Cys146 residues within this motif were crucial for MtUvrB function, Ser145 plays an important but less essential role. The SxSx motif-deleted mutant was drastically attenuated and three single (S143A, S145A and C146A) mutants and a double (S143A/S145A) mutant exhibited various degrees of severity in their DNA-binding, DNA helicase and ATPase activities. Taken together, these results highlight a hitherto unrecognized role for SxSx motif in the catalytic activities of UvrB.

Introduction

In all living organisms, robust biochemical and network-level mechanisms exist to ensure the integrity of their genomes. The nucleotide excision repair (NER) pathway is a frontline defense mechanism in all cells for repair of bulky DNA lesions that destabilises the DNA structure [[1], [2], [3]]. In bacteria, NER is an important DNA repair pathway that removes different types of structurally unrelated DNA adducts such as cyclobutane pyrimidine dimers, alkylation adducts, chemotherapeutic adducts as well as cisplatin interstrand crosslinks [[3], [4], [5]]. The process of NER involves the concerted action of six proteins, namely UvrA, UvrB, UvrC, DNA helicase II (UvrD), Pol I and DNA ligase, which orchestrate the sequential steps in the process of damage recognition, unwinding of DNA around the lesion, dual excision flanking the damaged site and finally, repair resynthesis and DNA ligation [[4], [5], [6], [7]]. Based on numerous genetic, biochemical and X-ray crystallographic studies, three major stages associated with the early steps of the NER pathway have been defined in the Escherichia coli paradigm [[3], [4], [5], [6], [7], [8], [9], [10], [11], [12]]. Notwithstanding the early uncertainties, a growing body of evidence suggests that the UvrA₂B₂ hetero-tetrameric complex is responsible for damage recognition [[10], [11], [12], [13], [14], [15]]. The ATP hydrolysis by (UvrA)₂ renders its dissociation from the lesion complex, thereby allowing UvrB₂ to recruit UvrC to form a pre-incision complex [[3], [4], [5], [6],[16], [17], [18], [19], [20], [21]]. The next step involves dual incisions by UvrC at sites flanking the DNA lesion, four/five nucleotides 3′ from the lesion and eight nucleotides from the 5′ side [[3], [4], [5], [6],[22], [23], [24], [25]]. The post-incision complex comprising of 12 to 13-mer single-stranded DNA and UvrBC subunits is removed by UvrD and Pol I [[3], [4], [5], [6],[25], [26], [27], [28]]. Finally, the gap is filled by DNA polymerase and the broken strands are sealed by DNA ligase [[3], [4], [5], [6],28].

The UvrB protein, which belongs to the superfamily 2 (SF2) of helicases, plays a central role in the NER pathway. A hallmark of the SF2 superfamily of helicases is the presence of seven conserved helicase motifs (motifs I, Ia, II, III, IV, V and VI), and some possess auxiliary domains [29,30]. These motifs are spread over two RecA-like domains that together constitute the helicase core, which by themselves couple ATP hydrolysis to DNA/RNA unwinding [29,30]. The multiple sequence alignment of MtUvrB revealed the existence of seven helicase motifs and also an auxiliary motif, downstream of the β-hairpin (Fig. 1A). One of the auxiliary domains is the SxSx motif, analogous to the TxGx motif of the SF2 superfamily of helicases (Fig. 1B). Various studies have found that TxGx motif is highly divergent and serine is often substituted for threonine [29,30]. The TxGx motif in some members of the SF2 superfamily of helicases is located in motif I [31]. The X-ray crystal structure of Bacillus caldotenax UvrB bound to DNA found that two serine residues (Ser141 and Ser143) in the SxSx motif, among others (Lys67, Ser91, Gln346 and Arg357), make electrostatic interactions with the inner strand of the duplex DNA (Fig. 1C) [32]. Here, we emphasize that these predictions have not been experimentally verified and, consequently, the significance of Ser141 and Ser143 residues in UvrB catalytic activities is not known.

To gain insights into the structure-function relationship of the SxSx motif, a molecular model was constructed for MtUvrB using the B. caldotenax UvrB X-ray crystal structure as a template (PDB code: 1D9X) and superimposed with B. caldotenax UvrB-DNA crystal structure (PDB code: 2FDC) [32,33]. The structures are highly similar (root mean square deviation of 1.37 Å, over the entire protein) and the DNA binding region is conserved. In addition to Ser143 and Ser145 residues in the MtUvrB SxSx motif, which track along the DNA, we found Cys146 (not seen in the B. caldotenax UvrB-DNA crystal structure) within the DNA binding region (Fig. 1D). To elucidate the functional roles of these residues in the SxSx motif, MtUvrB variants were constructed and examined with regards to their functional properties. Our results demonstrate markedly different degrees of severity in DNA binding, DNA helicase and ATPase activities. Taken together, these findings highlight a hitherto unrecognized role for SxSx motif in the catalytic activities of UvrB.