Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks.
Post-replication DNA repair facilitates the resumption of DNA synthesis upon replication fork stalling at DNA damage sites. Despite the importance of RAD18 and polymerase eta (Poleta) for post-replication repair (PRR), the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. We present evidence that human RAD18 complexed with RAD6B protein preferentially binds to forked and single-stranded DNA (ssDNA) structures, which are known to be localized at stalled replication forks. The SAP domain of RAD18 (residues 248-282) is crucial for binding of RAD18 complexed with RAD6B to DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at DNA damage sites in vivo and does not guide DNA Poleta to stalled replication forks. The SAP domain is also required for the efficient mono-ubiquitination of PCNA. The SAP domain mutant fails to suppress the ultraviolet (UV)-sensitivity of Rad18-knockout cells. These results suggest that RAD18 complexed with RAD6B is recruited to stalled replication forks via interactions with forked DNA or long ssDNA structures, a process that is required for initiating PRR.
Tsuji
Yuri
Y
Cell Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.
Watanabe
Kenji
K
Araki
Kimi
K
Shinohara
Masanori
M
Yamagata
Yuriko
Y
Tsurimoto
Toshiki
T
Hanaoka
Fumio
F
Yamamura
Ken-Ichi
K
Yamaizumi
Masaru
M
Tateishi
Satoshi
S
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D046912:Multiprotein Complexes; D018809:Proliferating Cell Nuclear Antigen; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C485954:RAD18 protein, human; C471546:UBE2B protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases