Biochemical characterization of AfPolX1: a X-family DNA Polymerase from Aspergillus fumigatus

Behera, Abhijit (2023) Biochemical characterization of AfPolX1: a X-family DNA Polymerase from Aspergillus fumigatus. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (Ms dissertation of Abhijit Behera (18MS010)) Thesis_18MS010.pdf - Submitted Version Restricted to Repository staff only Download (12MB)

Abstract

X-family DNA polymerases play an essential role in DNA repair. Although mammalian, especially human, DNA polymerases of this family have been extensively studied, X-family polymerases from other eukaryotes are yet to be well-characterized. We have investigated a novel X-family polymerase from the infectious fungus Aspergillus fumigatus Af293 (AfPolX1), annotated as a “Terminal deoxynucleotidyl Transferase” (TdT) in NCBI (Accession ID: XP_755218) and as a “DNA-directed DNA polymerase” in UniProt (Accession ID: Q4X1T5). We report that, unlike TdT, AfPolX1 has no template-independent nucleotide incorporation activity. Instead, it has single-nucleotide gap-filling activity, a characteristic feature of repair polymerases such as Pol μ. Our multiple sequence alignment (MSA) analysis revealed that AfPolX1 is most likely devoid of dRP lyase activity. Our biochemical characterization of AfPolX1 revealed that AfPolX1 is a template-dependent Pol μ with poor sugar selectivity and intermediate base selectivity. We have also performed in silico analysis of AfPolX2, another X-family polymerase from Aspergillus fumigatus Af293, with a Pol λ - like profile.

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