Coordination of dual incision and repair synthesis in human nucleotide excision repair

Abstract

Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5' incision by ERCC1-XPF precedes the 3' incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.

Recruitment and positioning determine the specific role of the XPF ‐ ERCC 1 endonuclease in interstrand crosslink repair

“…Nevertheless, our data indicate that the XE R670S mutant can still support NER to wild‐type levels. This is in line with previous data showing the human equivalent, hs XE R689A , can incise an NER substrate (Enzlin & Schärer, 2002; Staresincic et al , 2009; Su et al , 2012). Interestingly, the human mutant protein did show a shift in incision position, suggesting the residue is not directly involved in catalysis, but contributes to the proper orientation of the active site onto the DNA substrate (Su et al , 2012).…”

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

“…This open intermediate is framed by "Y-shaped" double-to single-stranded junctions, which constitute a preferred substrate for the structure-specific DNA endonucleases XPF and XPG [19,20]. The 5' incision by XPF-ERCC1 precedes the 3' incision by XPG [21]. Through double cleavage of 6 the damaged strand, the combined action of these two endonucleases leads to excision of the offending lesion in the form of an oligonucleotide segment of 24-32 residues [22,23].…”

“…Duplex integrity is reestablished by the action of DNA polymerases δ, ε and κ [24,25], in conjunction with RFC and PCNA. This DNA repair synthesis is carried out in line with the initial 5' incision, thus avoiding that excision of an oligonucleotide fragment causes the transient exposure of single-stranded DNA, which is at risk to be converted to a double-stranded break by inadvertent nuclease activity [21]. Finally, the newly synthesized repair patch is joined to the pre-existing strand by DNA ligase I and DNA ligase III [26,27].…”

Papel das proteínas XPD e DNA polimerase eta nas respostas de células humanas a danos no genoma

“…O mecanismo básico de ação do NER depende da ação orquestrada de forma sequencial de um grande número de proteínas (Volker et al, 2001;Costa et al, 2003), e consiste no reconhecimento da lesão, abertura da dupla hélice e excisão de um fragmento de 20 a 30 nucleotídeos contendo a região danificada da fita simples pela ação seqüencial de helicases e endonucleases (Staresincic et al, 2009) No GGR, o complexo XPC-hHR23B/RAD23B é o responsável pelo reconhecimento primário da lesão (Sugasawa et al, 1998), sendo capaz de reconhecer uma grande variedade de lesões que distorcem a fita dupla. XPC-hHR23B são os homólogos das proteínas Rad4 e Rad23 de leveduras; a proteína XPC purificada de extratos celulares pode ser coprecipitada com hHR23B, e na minoria das vezes com hHR23A (Masutani et al, 1994).…”

“…Após a clivagem pelo complexo XPF-ERCC1 já começa a ocorrer a síntese do novo fragmento de DNA antes que ocorra a clivagem a 3´ pela XPG, provavelmente de forma a minimizar a quantidade de DNA simples fita exposto durante o processo (Staresincic et al, 2009). …”