The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection

Abstract

We have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

“…; da Silveira et al. ). The assays of these targets are relatively fast, simple, and sensitive but not always fully informative because they only show the parasites that were proliferating at the time of harvest and not the parasites that were proliferating during a defined time period.…”

“…PCNA, MCM 2-7 , RPA) or the detection of protein modifications associated with the cell cycle (e.g. di-and trimethylation of lysine 76 in H3 histone) (Dang and Li 2011;Janzen et al 2006;Kumar et al 2009;da Silveira et al 2013). The assays of these targets are relatively fast, simple, and sensitive but not always fully informative because they only show the parasites that were proliferating at the time of harvest and not the parasites that were proliferating during a defined time period.…”

Nuclear export of replication protein A in the nonreplicative infective forms of Trypanosoma cruzi

“…Although being conserved in all eukaryotes, RPA heterotrimer has significant structural differences in trypanosomatids . Recently, we could show that, even with these differences, RPA from trypanosomatids presents its canonical functions, being implicated in DNA replication and DNA damage response .…”

Leishmania braziliensis replication protein A subunit 1: molecular modelling, protein expression and analysis of its affinity for both DNA and RNA

“…After sequencing of the gene and deducing of the encoded amino acid sequence, it was found that LaRPA-1, like the CfRPA-1 (see above), lacked the N-terminal RPA70N domain (present in human and yeast RPA-1), but shared with hRPA-1 and yRPA-1 a canonical N-terminal tRNA_anti domain, which is an OB (oligonucleotide/oligosaccharide-binding) fold structure [16]. More recently, these authors found evidence that the natural absence of RPA1N domain in LaRPA-1 does not impair its participation in DNA damage response and telomere protection [17].…”