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

Parasitology. 2013 Apr;140(4):547-59. doi: 10.1017/S0031182012002028. Epub 2013 Feb 7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Checkpoints / drug effects
  • DNA Breaks, Double-Stranded*
  • In Situ Nick-End Labeling
  • Leishmania / drug effects
  • Leishmania / genetics*
  • Leishmania / metabolism*
  • Nucleic Acid Synthesis Inhibitors / pharmacology
  • Phleomycins / pharmacology
  • Protozoan Proteins / metabolism*
  • Telomere / metabolism*

Substances

  • Nucleic Acid Synthesis Inhibitors
  • Phleomycins
  • Protozoan Proteins