Laboratory of Genomic Instability and Diseases

Scholastic record: (https://scholar.google.co.in/citations?user=fef1gNUAAAAJ&hl=en)

Genomic Instability and Diseases

Research Summary:
Replication of DNA strands during each cell division is a prerequisite and critical for proper function of any genome. It is critical not only because of the necessity to duplicate the genetic content, but also because faulty DNA synthesis renders spontaneous mutation, genome rearrangement, and breaks in DNA; and these are the ingredients of genomic instability. Genomic instability is directly associated with cell death, tumorogenesis and development of an array of complex diseases in humans; also virulence and multidrug resistance in pathogens. DNA polymerases (Pol) are the enzymes required for DNA synthesis virtually in all DNA transaction pathways; and malfunction or absence of these has been shown to be associated with diseases in humans. Therefore, we are accentuating to analyze functions of eukaryotic pols in following areas.
I) Genetic and biochemical studies in S. cerevisiae have indicated that the highly accurate and processive genomic DNA replication is carried out by the coordinated action of Polα-primase, Polδ and Polε. A number of mutants of Pol3 (catalytic subunit of Polδ) and Pol2 (catalytic subunit of Polε) causing mutagenesis have been genetically characterized; however, the biochemical basis and mechanism have not been yet investigated. In depth analysis of such mutants will unravel the mechanism and novel factors involved in mutagenesis in yeast and carcinogenesis in humans; and results obtained will be a basis to identify possible target molecules (/sites) for cancer therapy.
II) DNA replication does not proceed smoothly; as it encounters both DNA lesions and non-B form of DNA structures that must be by-passed to prevent collapsing of replication fork. Discoveries and subsequent studies on DNA pols which can replicate past an array of DNA damages with a very high degree of specificity in yeast and human have broadened our understandings on the mechanism of replication of damaged DNA (Trans-lesion DNA synthesis, TLS). However, these groups of pols synthesize undamaged DNA with low fidelity and efficiency; and thereby, often generate mutations. How does a cell manage to select the right polymerase for a specific function? Unlike TLS, the knowledge about replication of DNA containing unusual structure like hairpin, G-quartet, Z-DNA etc. is very limited. We are exploring the role of these alone or with associated factors like helicases in replicating DNA with secondary structures.
III) Candida albicans, an opportunistic pathogen, which can asymptomatically colonizes humans can cause severe infection in immunocompromised people or when the microbiome is imbalanced. Available evidences suggest that genomic instability in the form of mutations and chromosomal aberrations is a common occurrence in pathogenic and drug resistant clinical isolates of C. albicans, and is believed to be one of the critical determinants of its virulence. We hypothesize that mutagenesis due to replication defects not only it will adopt Candida albicans to survive against the odds of host defense mechanism but also mutations in certain genes would activate pathways of morphological transition and multidrug resistance. Thus we are engaged in characterizing DNA polymerases from C. albicans to decipher its role in DNA replication, mutagenesis and its association with morphogenesis and multidrug resistance. More recently we have shown that PCNA, the sliding clamp of DNA replication is functionally different from S. cerevisiae which can be effectively targeted against candidasis (http://www.biomedcentral.com/1471-2180/15/257) .

Genomic Instability and Disease

After joining ILS:

1. Kodavati Manohar and Narottam Acharya* (2015) Characterization of Proliferating Cell Nuclear Antigen (PCNA) from Pathogenic Yeast Candida albicans and its functional analyses in S. cerevisiae. BMC Microbiology, 15:257.
2. Yoon J.H+., Acharya N+., Jeseong P., Debashree Basu, Prakash S. and Prakash L. (2014) Identification of two functional PCNA binding domains in human DNA polymerase κ (+ joint first author). Genes to Cells., 19(7):594-601.
3. Acharya N., Kalssen R., Johnson R.E., Prakash L. and Prakash S. (2011) PCNA binding domains in all three subunits of yeast DNA polymerase δ modulate its function in DNA replication. Proc Natl Acad Sci USA., Nov.1; 108(44):17927-17932.

Book chapter

1. Narottam Acharya*, Kodavati Manohar, Shreenath Nayak, Avishek Chatterjee and Amrita Dalei (2106) DNA Polymerase: A putative drug target against Candidiasis. Frontiers in Life Sciences, Excel Indian Publishers, New Delhi, India. Pp. 12-23

During post-Ph.D.:
4. Acharya N+., Yoon J.H+., Hutwitz J., Prakash L. and Prakash S. (2010) DNA polymerase eta lacking the ubiquitin-binding domain promotes replicative lesion bypass in human cells (+ joint first author). Proc Natl Acad Sci USA., Jun 8; 107(23):10401-10405.
5. Acharya N., Johnson R.E., Pagès V., Prakash L. and Prakash S. (2009) Yeast Rev1 protein promotes complex formation of DNA polymerase zeta with Pol32 subunit of DNA polymerase delta. Proc Natl Acad Sci USA., Jun 16; 106(24):9631-9636.
6. Acharya N., Yoon J.H., Gali H., Unk I., Haracska L., Johnson R.E., Hutwitz J., Prakash L. and Prakash S. (2008) Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase eta in translesion DNA synthesis. Proc Natl Acad Sci USA., Nov 18; 105 (46):17724-17729.
7. Pagès V., Bresson A., Acharya N., Prakash L. Fuchs R.P. and Prakash S. (2008) Requirement of Rad5 for DNA polymerase zeta-dependent translesion synthesis in Saccharomyces cerevisiae. Genetics, 2008 Sep; 180(1):73-82.
8. Acharya N., Haracska L., Prakash S. and Prakash L. (2007) Complex formation of yeast Rev1 with DNA polymerase {eta}. Mol Cell Biol., Dec; 27 (23):8401-8408.
9. Acharya N., Brahma A., Haracska L., Prakash S. and Prakash L. (2007) Mutations in the Ubiquitin Binding UBZ Motif of DNA Polymerase {eta} Do Not Impair Its Function in Translesion Synthesis during Replication. Mol Cell Biol., Oct; 27(20):7266-7272.
10. Acharya N., Johnson R.E., Prakash S. and Prakash L. (2006) Complex formation with Rev1 enhances the proficiency of Saccharomyces cerevisiae DNA polymerase zeta for mismatch extension and for extension opposite from DNA lesions. Mol. Cell Biol., Dec. 26 (24), 9555-9563.
11. Acharya N., Haracska L., Johnson R.E., Unk I., Prakash S. and Prakash L. (2005) Complex formation of yeast Rev1 and Rev7 proteins: a novel role for the polymerase-associated domain. Mol. Cell Biol., Nov. 25 (21), 9734-9740.
12. Haracska L., Acharya N., Unk I., Johnson R.E., Hurwitz J., Prakash L. and Prakash S. (2005) A single domain in human DNA polymerase iota mediates interaction with PCNA: implications for translesion DNA synthesis. Mol. Cell Biol., Feb. 25 (3), 1183-1190

During Ph.D.:
13. Acharya N, Talawar R. K., Purnapatre K, Varshney U. (2004) Use of sequence microdivergence in mycobacterial ortholog to analyze contributions of the water-activating loop histidine of Escherichia coli uracil-DNA glycosylase in reactant binding and catalysis. Biochem. Biophys. Res. Commun., 320(3), 893-899.
14. Acharya, N., Talawar R. K., Saikrishnan, K., Vijayan, M and Varshney, U. (2003) Substitutions at tyrosine 66 of Escherichia coli uracil DNA glycosylase lead to characterization of an efficient enzyme that is recalcitrant to product inhibition. Nucleic Acids Res., 31(24), 7216-7226.
15. Acharya, N., Kumar P. and Varshney, U. (2003) Complexes of Uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases. Microbiology 149, 1647-1658.
16. Saikrishnan, K., Jeyakanthan, Venkatesh, J., Acharya, N., Purnapatre, K., Sekar, K., Varshney, U. and Vijayan, M. (2003) Structure of Mycobacterium tuberculosis single stranded DNA binding protein. Variability in quaternary structure and its implications. J. Mol. Biol., 331, 385-393.
17. Acharya, N., Roy, S., and Varshney, U. (2002) Mutational analysis of the uracil DNA glycosylase inhibitor protein and its interaction with Escherichia coli uracil DNA glycosylase. J. Mol. Biol., 321 (4), 579-590.
18. Acharya, N., and Varshney, U. (2002) Biochemical properties of single stranded DNA binding protein from Mycobacterium smegmatis, a fast growing mycobacterium and its physical and functional interaction with uracil DNA glycosylases. J. Mol. Biol., 318 (5), 1251-1264.
19. Handa, P.+, Acharya, N.+, and Varshney, U. (2002) Effects of mutations at tyrosine 66 and asparagine 123 in the active site pocket of Escherichia coli uracil DNA glycosylase on uracil excision from synthetic DNA oligomers: evidence for the occurrence of long-range interactions between the enzyme and substrate. (+ joint first author) Nucleic Acids Res, 30(14), 3086-3095.
20. Handa, P., Acharya, N., Talwar R. K., Roy, S., and Varshney, U. (2002) Contrasting effects of mutating active site residues, aspartic acid 64 and histidine 187 of Escherichia coli uracil-DNA glycosylase on uracil excision and interaction with an inhibitor protein. Indian J. Biochem. Biophys., 29, 312-317.
21. Saikrishnan, K., Jeyakanthan, Venkatesh, J., Acharya, N., Purnapatre, K., Sekar, K., Varshney, U. and Vijayan, M. (2002) Crystallization and preliminary X-ray studies on the single stranded DNA binding protein from Mycobacterium tuberculosis. Acta Crystallogr D Biol Crystallogr, 58, 327-329.
22. Handa, P., Acharya, N. and Varshney, U. (2001) Chimeras between single stranded DNA binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil-DNA glycosylases. J. Biol. Chem. 276, 16992-16997.
23. Handa, P., Acharya, N., Thanedar, S., Purnapatre, K. and Varshney, U. (2000) Distinct properties of single stranded DNA binding protein from Mycobacterium tuberculosis and its functional characterization in Escherichia coli. Nucleic Acids Res. 28, 3823-3829.
24. Sadhale, P., Sharma, N., Beena, P, Katoch, A., Acharya, N., and Singh, S. K. (1998) Modulation of polymerase II composition: a possible mode of transcriptional regulation of stress response in eukaryotes. J. Biosci. 23, 331-335.

Genomic Instability and Diseases

Ph.D. students: 

K. Manohar (CSIR – SRF) (kodavati31@yahoo.co.in) DNA Replication in C. albicans

A. Chatterjee (DBT -JRF) (avishek1809@gmail.com)  DNA Replication in S. cerevisiae

P. Khandagale (DBT -JRF) (prashant.khanda009@gmail.com) DNA Replication in human

Lab Technicians:

Sitendra Panda (sitendrails2013@gmail.com )

 Amrita Dalai (amritadalei07@yahoo.co.in ) XPR Facility

Our Alumini:

 S. Nayak, Ph.D. (DBT-RA) (shreenathnayak@gmail.com )

Jawed Alam, Ph.D. (CSIR-RA) (jawedalam81@yahoo.com )

Also see

Genomic Instability and Diseases

Ongoing sponsored Research Projects:

1. “Characterization of pol3-t mutant of yeast DNA polymerase δ and its role in mutagenesis”.  Funded by CSIR (2014-2017)

2. “Understanding the role of DNA polymerase eta dependent trans-lesion DNA synthesis in pathogenicity of Candida albicans”. Funded by DBT (2116-2019)

3.  “Deciphering the role of DNA polymerase delta dependent mutagenesis in   morphogenesis, multidrug resistance and fungal pathogenesis of C. albicans.” Funded by SERB-DST (2016-2019)