Work done in the lab of Prof. K. Muniyappa, Professor, at Department of Biochemistry, Indian Institute of Science, Bangalore and Prof. K. D. Sonawane at Structural Bioinformatics Unit, Shivaji University, Kolhapur, India
About author
Manoj Thakur obtained his bachelor’s honors degree from the Botany Department, Acharya Narendra Dev College, University of Delhi, South Campus, New Delhi. He earned a Master’s degree in Life Sciences, at the School of Life Sciences, Jawaharlal Nehru University, New Delhi, where he got his early exposure to Molecular Biology in the lab of Prof. Rohini Muthuswami. Subsequently, he joined the laboratory of Prof. K. Muniyappa at the Biochemistry Department, Indian Institute of Science (IISc), Bangalore for his Ph.D. work in the field of DNA repair. After receiving his Ph.D. degree, he moved to Memorial Sloan Kettering Cancer Center, New York for a year and returned as a Research Associate back at the Biochemistry Department, IISc in the laboratory of Prof. K. Muniyappa. His research interest lies in identifying the role(s) of proteins involved in the genome maintenance of mycobacteria that have the potential as drug targets to mitigate the current crisis due to the emergent drug-resistant strains.
Interview
How would you explain your research outcomes to the non-scientific community?
The maintenance of DNA integrity is a crucial task for the proper functioning of the cell. However, the physicochemical constitution of DNA is constantly assaulted by a perplexing diversity of lesions that arise from physical or chemical attacks from the environment, byproducts of normal cellular metabolism, and errors during replication (Figure 1). These damaging agents produce lesions such as modified nucleotides, bulky adducts on nucleotides, single- and double-strand breaks, inter- or intra-strand crosslinks, apurinic or apyrimidinic base free sites, mismatches, deletions, and insertions (Figure 1). If left unrepaired, these lesions ultimately lead to genomic/gene instability, compromise cellular growth or replication, and various diseases like cancer, neurological disorders, immunodeficiency, Fanconi anemia in humans, premature aging, etc.
Evolution has invested significantly by implementing an intricate network of DNA repair systems in every organism — from bacteria to humans —to circumvent the diverse and generally adverse outcome(s) of DNA damage. These different DNA repair systems can be classified on the basis of the type of lesion being processed and protein components or mechanisms involved in the repair pathway. In other words, a cell deploys an arsenal of complementary protein molecular machinery that recognizes a class of DNA lesions and targets them for removal from the DNA. In the Escherichia coli paradigm, these include direct reversal of damage, nucleotide excision repair (NER), mismatch repair (MMR), base excision repair (BER), homologous recombination (HR) mediated repair, and non-homologous end joining (NHEJ) (Figure 1). These studies had also been recognized with “Nobel Prize in Chemistry 2015” to Tomas Lindahl, Paul Modrich, and Aziz Sancar highlighting the significance of DNA damage and repair systems in all three domains of life.
NER system is a multi-step, ATP-dependent process and catalytically versatile because of its capacity to remove myriad types of chemically and structurally diverse DNA lesions. Albeit with variable efficiencies, NER is critical for the removal of UV-induced photoproducts (cyclobutane dimers, 6–4 photoproducts, and thymine glycol), bulky adducts, apurinic or apyrimidinic (AP) sites, nicks, gaps, and cross-links. NER pathway has been characterized to an unprecedented level in bacteria, yeast, and humans and these studies envisage six discrete steps: DNA scanning, damage detection, damage verification, incision, removal of damage, DNA synthesis, and ligation as depicted in Figure 1. In the eubacterial domain, the pathway commences when the UvrA2B2 heterotetrameric complex bound to ATP scans the DNA for lesions by utilizing energy from ATP hydrolysis. After the damage is recognized by the UvrA component, the lesion is then occupied by UvrB for damage verification and UvrA gets evicted in an ATP hydrolysis dependent manner. Specifically, UvrA detects damage-induced structural distortions in the DNA, and then the lesion-containing strand is transferred to the active site of UvrB whereby it is embraced by the beta-hairpin motif for damage verification, resulting in a tight UvrB-preincision complex. While doing so, UvrB arrives at the damaged site by virtue of its ability to translocate on single-stranded DNA (ss-DNA) and unwind double-stranded DNA (dsDNA). Thus, UvrC is recruited by UvrB-preincision complex precisely on the damaged strand, where it specifically cleaves at 4-5 nucleotides 3ʹ and 8 nucleotides 5ʹ to the damage site in a concerted manner. Subsequently, the UvrD helicase removes the lesion-containing short oligonucleotide while UvrB remains bound to the gapped DNA until it is displaced by the DNA PolI mediated non-conservative DNA synthesis. Finally, the reaction is faithfully completed by DNA LigA that seals the nick, and the damaged site is restored back to the undamaged state.
Although much has been understood about bacterial NER proteins by eliciting information from structural, biochemical, and combination of genetic or single molecule studies, little is known about the UvrC protein perhaps due to its inactivity or instability in vitro. Since its first purification by Aziz Sancar in 1981, studies from the past four decades have highlighted the fact that UvrC is a multi-domain protein that contains N-terminal GIY-YIG endonuclease domain, cysteine-rich module, UvrBC motif, C-terminal RNase H endonuclease domain and helix-hairpin-helix (HhH)2 motif (Figure 2). Notably, molecular determinants responsible for 3ʹ and 5ʹ incision reactions are present on the catalytic GIY-YIG and RNase H endonuclease domains, respectively. Unlike the NER proteins of eukaryotes, studies with UvrA, UvrB, and UvrC subunits are confined more or less to their participation in the bacterial NER pathway. There is nascent evidence for functional crosstalk between NER, HR and DNA replication; however, the catalytic mechanisms are barely understood. A particularly significant example from the literature is the functional crosstalk between NER and HR in the recognition and removal of DNA interstrand crosslinks (ICLs). Thus, many proteins that are identified as having key roles in NER also function in DNA replication and HR in eukaryotic cells. In line with these observations, a notable contribution from Noora Goosen’s laboratory demonstrated that whilst E. coli uvrA, uvrB, and uvrD are needed in polA deleted cells for the replication backup system, uvrC suppresses this alternative DNA replication pathway, clearly suggesting interplay between NER and DNA replication. In this study, we interrogated the substrate specificity landscape of UvrC and demonstrated an unanticipated mechanism underlying UvrC-mediated suppression of DNA replication.
How do these findings contribute to your research area?
Understanding the structure and functions of proteins involved in the survival and maintenance of mycobacterial genome is fundamental to advance their biomedical potential. Herein, we report proof-of-principle experiments demonstrating that E. coli and M. tuberculosis UvrC exhibit high specificity and affinity toward a wide range of complex, branched DNA structures that are obligatory intermediates of DNA replication/HR, in contrast to the commonly used model NER substrates. More importantly, we provided first biochemical evidence that the structure-specific DNA binding activity of UvrC strongly correlates with the efficiency with which it cleaves various branched DNA species via its DNA-independent ATPase activity. In-silico analyses guided site-specific mutations revealed that amino acid residues Glu595 and Arg597 in the MtUvrC (HhH)2 motif are essential for its Holliday junction (a DNA structure intermediate formed during HR pathway) binding activity. We further demonstrated that by its ability to hydrolyze ATP, M. tuberculosis UvrC inflicts multiple nicks on each strand of these various DNA replication/recombination intermediates. Invoking Occam’s razor and underscoring a broad biological significance, MtUvrC as an ATP dependent endonuclease might be used by cells to destroy the aberrant, persistent branched DNA species formed during DNA replication and HR.
“We provided first biochemical evidence that the structure-specific DNA binding activity of UvrC strongly correlates with the efficiency with which it cleaves various branched DNA species via its DNA-independent ATPase activity.”
What was the exciting moment during your research?
As described earlier, UvrC was extensively characterized with respect to its dual incisions bracketing the lesion. In the light of previous literature, we were curious to check various activities of the MtUvrC by asking a very simple fundamental question to what extent the mechanistic model of E. coli (EcUvrC) could be applicable in M. tuberculosis? Systematic analysis of substrate specificity with all the DNA replication intermediates surprised us with the observation that MtUvrC preferred HJ and replication fork over canonical lesions containing ds-DNA. Although DNA binding does not dictate the physiological relevance and given its unique substrate specificity, we next asked whether it partakes in the processing of DNA replication/HR intermediates. We were thrilled to observe multiple nicks facilitated by MtUvrC on multiple types of branched DNA species in the absence of any lesion and specifically in the presence of ATP (Figure 2). The second exciting moment was when I approached Prof. K.D. Sonawane for the collaboration and without any delay he started supervising the homology modelling, molecular docking, and simulation studies (performed by Rishikesh S Parulekar and Sagar S Barale). These studies further validated the involvement of key amino acid residues of MtUvrC in the HJ binding, ATPase, and endonuclease activity.
What do you hope to do next?
The current study of UvrC along with our other studies utilizing UvrA, and UvrB subunits of M. tuberculosis, developed a biochemical framework for the screening of anti-tubercular agents. Exploring the therapeutic potential of the UvrABC excinuclease complex is another challenging task and I do hope to conduct the inhibitor screening in near future.
Where do you seek scientific inspiration from?
For early career scientists like me or other people interested in getting settled into the research field, I realize personally that perseverance, and determination towards a particular question of interest or any task in conjunction with ethics are the absolute requirements to succeed in science. A lesson learned from K. Muniyappa deserves a special mention here. During my six years of Ph.D., I was glad to behold the working hours of Prof. K. Muniyappa usually from 8:30 AM to 7 PM every day except for Sunday when he would only work “a half a day”. I still wonder but never asked what he does for the rest of the day? In the halo of his extreme brilliance, not only myself but all his other students were inspired and worked systematically to a higher level and met his expectations. However, we all are very familiar with the gradual nature of progress in each experiment, and the fear of artifact generation, indeed, a frustrating part of science. In all these hectic and gloomy situations, opportunities to have stimulating scientific or problem-solving discussions with bright friends within or outside the department, and of course, with the scientific community inspired me a lot. Thanks to technology, google, the internet, and social media that connect people all throughout the globe!
How do you intend to help Indian science improve?
I highly acknowledge all major funding bodies of India for the support they provided for conducting my own research. Despite this, I personally feel that amendments are needed in the current framework of allocating funds that are responsible for running Indian science labs nationwide. Moreover, it is not a question of how I can improve Indian science. The real question should be how we all could improve Indian science. We all must appreciate that science cannot be done without money and in a cocoon. If much of the funds being started were allocated to every individual lab, all the recruits would get a chance to show their real talent. In addition to this economic attribute, one must establish extensive or faithful collaborations within the country or abroad for greater understanding or tackling research questions being addressed via different ideas or technology exchange.
Reference
Thakur M, Parulekar RS, Barale SS, Sonawane KD, Muniyappa K. Interrogating the substrate specificity landscape of UvrC reveals novel insights into its non-canonical function. Biophys J. 2022 Jul 9:S0006-3495(22)00558-6. doi: 10.1016/j.bpj.2022.07.012. Epub ahead of print. PMID: 35810330. (https://pubmed.ncbi.nlm.nih.gov/35810330/)
Copy Editor: Nikita Nimbark