Biology subjects

Chadda, A.

Publications and source records attributed to Chadda, A..

5 recordsLinked to original sources

bioRxiv2026

Bulky lesions on the displaced strand stimulate DNA unwinding by dimeric UvrD-family helicases

UvrD-family SF1A helicases play a variety of biological roles in DNA metabolism including replication, recombination, DNA repair, and conjugative plasmid transfer. The family is described by a subdomain architecture consisting of two RecA motor subdomains (1A and 2A) that each contain an auxiliary B-domain insertion (1B and 2B). Monomeric UvrD-family enzymes possess ATPase and 3-5 single-stranded DNA translocase activity but lack helicase activity. An enzyme dimer formed through a 2B-2B domain interface acts as a processive DNA helicase. A partial explanation for this observation, based on structural comparison between monomeric and dimeric DNA bound complexes, is that dimerization removes inhibitory contacts between the 2B domain and the double-stranded DNA, thus activating the helicase. However, biochemical observations reveal additional aspects of the dimeric mechanism that cannot solely be explained by movement of the 2B subdomain. Here, we present data showing that the Mycobacterium tuberculosis UvrD1 dimer interacts with the displaced strand of the duplex (i.e., the 5-3 strand in the direction of unwinding). In particular, bulky lesions on the displaced strand-including a thymine dimer-led to more efficient unwinding over a finite range of duplex lengths. These findings suggest a model for dimeric UvrD-family unwinding wherein one subunit makes intimate contacts with the displaced strand and that this interaction promotes the processive DNA unwinding unique to dimers.

biophysics↗

bioRxiv2026

A Conserved Mechanism for Dimerization and Activation of Superfamily 1A UvrD-family Helicases

DNA helicases are ATP-dependent motor proteins that catalyze duplex DNA unwinding and are involved in DNA repair, recombination and replication restart. Prominent members of the non-hexameric SF1A UvrD-family helicases are E. coli UvrD, Rep, B. stearothermophilus PcrA and M. tuberculosis UvrD1. SF1A monomers are processive 3 to 5 single stranded DNA translocases, but need to be activated to become DNA helicases. One mechanism of activation is dimerization. Whereas Rep, UvrD and PcrA form non-covalent dimers, the Mtb UvrD1 helicase forms a redox-dependent covalent dimer. Dimerization of Mtb UvrD1 occurs between the same regulatory domain (2B) within each subunit stabilized by a disulfide bond formed between the same cysteine (Cys451) within each subunit. Dimerization relieves an inhibitory interaction between the 2B domain and duplex DNA within the monomer-DNA complex. We show here that Rep, UvrD and PcrA dimerize using the same 2B-2B interface. By placing a Cys residue within the 2B domains of Rep, UvrD and PcrA in the structurally equivalent position occupied by Cys451 of Mtb UvrD1, all three enzymes form redox-dependent covalent dimers that are constitutively active helicases with increased processivity compared to the non-covalent dimers. Hence, the 2B domain is a general dimerization domain for UvrD-family SF1A helicases.

biochemistry↗

bioRxiv2024

Structural Basis for Dimerization and Activation of UvrD-family Helicases

UvrD-family helicases are superfamily 1A motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD family monomers translocate along single stranded (ss) DNA but need to be activated by dimerization to unwind DNA in the absence of force or accessory factors. However, prior structural studies have only revealed monomeric complexes. Here, we report the first structures of a dimeric UvrD-family helicase, Mycobacterium tuberculosis UvrD1, both free and bound to a DNA junction. In each structure, the dimer interface occurs between the 2B subdomains of each subunit. The apo UvrD1 dimer is observed in symmetric compact and extended forms indicating substantial flexibility. This symmetry is broken in the DNA-bound dimer complex with leading and trailing subunits adopting distinct conformations. Biochemical experiments reveal that the E. coli UvrD dimer shares the same 2B-2B interface. In contrast to the dimeric structures, an inactive, auto-inhibited UvrD1 DNA-bound monomer structure reveals 2B subdomain-DNA contacts that are likely inhibitory. The major re-orientation of the 2B subdomains that occurs upon UvrD1 dimerization prevents these duplex DNA interactions, thus relieving the auto-inhibition. These structures reveal that the 2B subdomain serves a major regulatory role rather than participating directly in DNA unwinding.

biochemistry↗

bioRxiv2023

Mycobacterium tuberculosis Ku stimulates multi-round DNA unwinding by UvrD1 monomers

Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to infection, the bacterium is exposed to both reactive oxygen species and nitrogen intermediates that can cause DNA damage. It is becoming clear that the DNA damage response in Mtb and related actinobacteria function via distinct pathways as compared to well-studied model bacteria. For example, we have previously shown that the DNA repair helicase UvrD1 is activated for processive unwinding via redox-dependent dimerization. In addition, mycobacteria contain a homo-dimeric Ku protein, homologous to the eukaryotic Ku70/Ku80 dimer, that plays roles in double-stranded break repair via non-homologous end-joining. Ku has been shown to stimulate the helicase activity of UvrD1, but the molecular mechanism, as well as which redox form of UvrD1 is activated, is unknown. We show here that Ku specifically stimulates multi-round unwinding by UvrD1 monomers which are able to slowly unwind DNA, but at rates 100-fold slower than the dimer. We also demonstrate that the UvrD1 C-terminal Tudor domain is required for the formation of a Ku-UvrD1 protein complex and activation. We show that Mtb Ku dimers bind with high nearest neighbor cooperativity to duplex DNA and that UvrD1 activation is observed when the DNA substrate is bound with two or three Ku dimers. Our observations reveal aspects of the interactions between DNA, Mtb Ku, and UvrD1 and highlight the potential role of UvrD1 in multiple DNA repair pathways through different mechanisms of activation. Declaration of Interests None

biophysics↗

bioRxiv2021

Mycobacterium tuberculosis DNA-repair helicase UvrD1 is activated by redox-dependent dimerization via a conserved 2B domain cysteine.

Mycobacterium tuberculosis (Mtb) causes Tuberculosis and, during infection, is exposed to reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric, however it is well-known that E. coli UvrD and other UvrD-family members exhibit monomer-dimer equilibria and unwind as dimers in the absence of accessory factors. Here, we reconcile these incongruent studies by showing that Mtb UvrD1 exists in monomer, dimer, and tetramer oligomeric forms where dimerization is regulated by redox potential. We identify a 2B domain cysteine, conserved in many Actinobacteria, that underlies this effect. We also show that UvrD1 DNA unwinding activity correlates specifically with the dimer population and is thus titrated directly via increasing positive (i.e. oxidative) redox potential. Consistent with the regulatory role of the 2B domain and the dimerization-based activation of DNA unwinding in UvrD-family helicases, these results suggest that UvrD1 is activated under oxidizing conditions when it may be needed to respond to DNA damage during infection.

biochemistry↗