Site Directed Mutagenesis Reveals Functional Importance of Conserved Amino Acid Residues Within the N-terminal Domain of Dpb2 in Budding Yeast

Overview

Journal Arch Microbiol

Specialty Microbiology

Date 2024 Dec 17

PMID 39690285

Authors

Huma Shaz

Prakash Nandi

Sugopa Sengupta

Affiliations

Soon will be listed here.

Abstract

In spite of being dispensable for catalysis, Dpb2, the second largest subunit of leading strand DNA polymerase (Polymerase ε) is essential for cell survival in budding yeast. Dpb2 physically connects polymerase epsilon with the replicative helicase (CMG,Cdc45-Mcm-GINS) by interacting with its Psf1 subunit. Dpb2-Psf1 interaction has been shown to be critical for incorporating polymerase ε into the replisome. Site-directed mutagenesis studies on conserved amino acid residues within the N-terminal domain of Dpb2 led to identification of key amino acid residues involved in interaction with Psf1 subunit of GINS. These amino acid residues are found to be well conserved among Dpb2 orthologues in higher eukaryotes thereby indicating the protein-protein interaction to be evolutionarily conserved. Replicating cells are known to mount a strong replicative stress response and DNA damage response upon exposure to diverse range of stressors. Here, we show that the absence of the N-terminal domain of Dpb2 increases the vulnerability of the budding yeast cells towards the cytotoxic effects of hydroxyurea (HU) and methyl methane sulphonate (MMS). Our results illustrate the importance of N-terminal domain of Dpb2 not only during replisome assembly but also in coordinating stress response in budding yeast. Considering high degree of sequence conservation across eukaryotes, Dpb2 subunit of leading-strand DNA polymerase appears to have important implications in maintenance of genome integrity.

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