Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi

Abstract

Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi.

Replication Protein A‐1 Has a Preference for the Telomeric G‐rich Sequence in Trypanosoma cruzi

“…We have previously shown that TcRPA‐1 interacts with ssDNA, while TcRPA‐2 does not bind ssDNA in an in vitro assay (Pavani et al. ). Since the termini of chromosomes expose the G‐overhang single‐stranded telomeric repeats, we first analyzed the abilities of TcRPA‐1 and TcRPA‐2 to bind the telomeric single‐stranded DNA.…”

“…Samples were incubated with mouse anti‐RPA‐1 antibody (Pavani et al. ) or with preimmune serum under agitation for 2 h at 4 °C. Samples were then harvested and transferred to a new tube containing 40 μl of protein G Sepharose beads (GE Healthcare Life Sciences, Amersham Place, U.K.) previously incubated with 5 mg/ml BSA for 2 h at 4 °C under agitation.…”

“…Recombinant TcRPA‐1 and TcRPA‐2 were expressed and purified as described in (Pavani et al. ). For EMSA assays, the single‐stranded DNA oligonucleotides used in these experiments were labeled with DIG‐11‐ddUTP using a Dig Gel Shift kit (Roche, Welwyn Garden City, Hertfordshire, U.K.).…”

“…The in silico model of TcRPA‐1‐OBF12 was obtained by our group previously (Pavani et al. ) by the profile‐based threading method program Phyre2 (Kelley et al. ) and is available at the Model Archive database with the access code ma‐azgkp.…”

“…The Trypanosoma cruzi RPA homolog was recently described and presents essential functions such as participation in replication and DNA damage response (Pavani et al. ). However, its interaction with telomeres remains to be elucidated.…”