title: Biochemical studies with DNA polymerase beta and DNA polymerase beta-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance.
abstract: Mammalian DNA polymerase beta is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerasebeta, TcPolbeta and TcPolbetaPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolbetaPAK, in comparison to TcPolbeta, conducted DNA synthesis over a much broader pH range. TcPolbeta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolbeta were not observed for TcPolbetaPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolbeta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.
authors.affiliation: Departamento de Bioquímica e Imunologia, ICB/UFMG, Brazil.
authors.forename: Débora de Oliveira
authors.initials: Dde O
authors.lastname: Lopes
authors.forename: Bruno Luiz Fonseca
authors.initials: BL
authors.lastname: Schamber-Reis
authors.forename: Carlos Gustavo
authors.initials: CG
authors.lastname: Regis-da-Silva
authors.forename: Matheus Andrade
authors.initials: MA
authors.lastname: Rajão
authors.forename: Wanderson Duarte
authors.initials: WD
authors.lastname: Darocha
authors.forename: Andréa Mara
authors.initials: AM
authors.lastname: Macedo
authors.forename: Glória Regina
authors.initials: GR
authors.lastname: Franco
authors.forename: Sheila Cristina
authors.initials: SC
authors.lastname: Nardelli
authors.forename: Sérgio
authors.initials: S
authors.lastname: Schenkman
authors.forename: Jean-Sébastien
authors.initials: JS
authors.lastname: Hoffmann
authors.forename: Christophe
authors.initials: C
authors.lastname: Cazaux
authors.forename: Sérgio Danilo Junho
authors.initials: SD
authors.lastname: Pena
authors.forename: Santuza Maria Ribeiro
authors.initials: SM
authors.lastname: Teixeira
authors.forename: Carlos Renato
authors.initials: CR
authors.lastname: Machado