Indian Journal of Virology
  • Year: 2006
  • Volume: 17
  • Issue: 2

S.43. Isolation and functional characterization of replication protein A (RPA) in geminiviral replication

  • Author:
  • Dharmendra K. Singh, Nirupam Roy Choudhury, Sunil K. Mukherjee
  • Total Page Count: 2
  • Page Number: 123 to 124

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067.

Abstracts of the papers presented at the 16th Annual Convention and International Symposium of Indian Virological Society on “Management of Vector-Borne Viruses” at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Hyderabad, India, February 7–10, 2006.

Abstract

Mungbean yellow mosaic India virus (MYMIV) belongs to the family of Geminiviridae and comprises of bipartite, single stranded, circular genome(s) of about 2.7 kb. It replicates inside the nucleus of the infected host cell via rolling circle (RCR) mode of DNA replication. It encodes only few replication proteins of its own, the major one being Replication initiator protein (Rep) and interplay with the host cellular machinery for complete replication of its genome. Here the role of single stranded DNA binding protein Replication protein A (RPA) of the host in DNA replication of MYMIV was examined. The two RPA subunits were isolated from pea, namely the RPA70 kDa and RPA32 kDa using RACE-PCR approach and the genes were functionally characterized by complementation assay as well as yeast two hybrid interaction between RPA32 and RPA70 kDa subunits. The roles of the two subunits were validated in a yeast system which could support MYMIV DNA replication and further confirmed by plasmid retention assay in the temperature sensitive mutant of yeast for RPA32 and RPA70. We present the evidences that only RPA32 kDa subunit directly interacted with MYMIV-Rep both in vitro, by applying pull down assay, as well as in yeast two-hybrid system. A novel interacting site present at the C- terminus of the Rep was responsible for this interaction. RPA32 also modulated the functions of Rep by enhancing its ATPase activity and down regulating its nicking and closing activities. Our data suggest the role of RPA32 subunit in RCR mode of DNA replication and established that RPA may be involved at the elongation stage of MYMIV DNA replication.