Professor
Fellow of National Academy of Sciences

email: ss5gp@yahoo.co.in, ssaha@south.du.ac.inphone: 011-24157380

Area of research: DNA replication and chromatin modifications

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Research description:

Our laboratory researches two aspects of DNA biology: DNA replication and chromatin modifications. The two model systems being investigated are the protozoan parasite Leishmania donovani (causative agent of kala azar) and the euryarchaeon Picrophilus torridus.

Eukaryotic DNA replication is a largely conserved process where the synthesis of DNA in the S phase of the cell cycle is preceded by the licensing of origins in the G1 phase. The process of licensing of origins begins with the assembly of pre-replication complexes (pre-RCs) at or very near origins in the G1 phase.  Multiple proteins form the pre-RCs, starting with the association of the Origin Recognition Complex (ORC, comprising Orcs1-6) with origin DNA, followed by the sequential recruitment of Cdc6, Cdt1, and the MCM complex (comprising Mcm2-7), among other proteins. The association of the MCM complex licenses origins to fire, and as cells enter S phase, specific components of the complex are phosphorylated and additional proteins like Cdc45 and GINS are recruited to form the pre-initiation complex, thus allowing replication to ensue upon the association of the replicative polymerases.  Based on genome sequence annotation it appears that the process in Leishmania is broadly conserved with other eukaryotes, with orthologs of several of the pre-RC and pre-IC being identified. However, several of the conserved orthologs are absent, reflecting the early divergence of Leishmania from the eukaryotic lineage.  Thus, while Orc1 and Orc4 have been identified, Orcs 2,3 5 and 6 are missing.  No Cdt1 has been identified either. Our laboratory has charactered three proteins: Orc1, Mcm4, and PCNA, and is currently characterizing Cdc45.

The second aspect our lab has been investigating is histone modifications. In general, eukaryotic histones are highly conserved in sequence and the post-translational modifications (PTMs) they carry. These include specific acetylation, methylation, phosphorylation, and ubiquitination events to name a few. These modifications regulate a myriad of cellular processes such as transcription, replication and DNA repair. However, trypanosomatid histones are divergent in sequence from histones of other eukaryotes, and consequently, the modifications they carry are also divergent.  While no information is available regarding the global landscape of histone modifications in Leishmania species, work from the closely related Trypanosoma species have identified several modification marks.  Our lab has examined histone acetylation events in Leishmania donovani. Using biochemical assays we have identified three histone H4 acetylation marks in vitro, and using the approach of creating genomic knockouts we have validating two of these in vivo as well.  By characterizing the phenotypes of the knockout lines we determined that while histone acetyltransferase HAT3 acetyated histone H4 at the K4 position, and played a role in modulating the cell’s response to UV-induced DNA damage, histone acetyltransferase HAT2 acetyated histone H4 at the K10 position and modulated gene expression, most likely through the acetylation of promoter regions. Histone acetyltransferase HAT4 was found to modulate events at the G2/M phase of the cell cycle. We are currently beginning investigations into histone methylation events.

Select Publications:

Saha, S. (2020). Histone modifications and other facets of epigenetic regulation in trypanosomatids: leaving their mark. mBio 11:e01079-20.

Yadav, A, Sharma, V, Pal, J, Gulati, P, Goel, M, Chandra, U, Bansal, N, Saha, S. (2020). DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival. PLoS Pathogens. May 15;16(5):e1008190.

Chandra, U, Yadav, A, Kumar, D, and Saha, S. (2017).  Cell cycle stage-specific transcriptional activation of cyclins mediated by HAT2-dependent H4K10 acetylation of promoters in Leishmania donovani. PLoS Pathogens 13(9): e1006615

Kumar, D, and Saha, S. (2015). HAT3-mediated acetylation of PCNA precedes PCNA monoubiquitination following exposure to UV radiation in Leishmania donovani. Nucleic Acids Res. 43(11):5423-41

Arora, J, Goswami, K, and Saha, S. (2014). Characterization of the replication initiator Orc1/Cdc6 from the archaeon Picrophilus torridus. J Bacteriol. 196: 276-286.