Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair

DNA Repair (Amst). 2014 Dec;24:46-56. doi: 10.1016/j.dnarep.2014.09.013. Epub 2014 Oct 16.

Abstract

The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.

Keywords: DNA damage; DNA repair; DNA replication; Nucleotide excision repair; RPA; Replication stress.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle
  • Cell Line / radiation effects
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • DNA Damage / radiation effects
  • DNA Repair*
  • DNA Replication
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Kinetics
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Replication Protein A / genetics
  • Replication Protein A / metabolism*
  • Replication Protein C / genetics
  • Replication Protein C / metabolism
  • Ultraviolet Rays

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Proliferating Cell Nuclear Antigen
  • RFC1 protein, human
  • RPA1 protein, human
  • Replication Protein A
  • xeroderma pigmentosum group F protein
  • Green Fluorescent Proteins
  • Replication Protein C