A fidelity mechanism in DNA polymerase lambda promotes error-free bypass of 8-oxo-dG.

8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxo-dG) has high mutagenic potential as it is prone to mispair with deoxyadenine (dA). In order to maintain genomic integrity, post-replicative 8-oxo-dG:dA mispairs are removed through DNA polymerase lambda (Pol λ)-dependent MUTYH-initiated base excision repair (BER). Here, we describe seven novel crystal structures and kinetic data that fully characterize 8-oxo-dG bypass by Pol λ. We demonstrate that Pol λ has a flexible active site that can tolerate 8-oxo-dG in either the anti- or syn-conformation. Importantly, we show that discrimination against the pro-mutagenic syn-conformation occurs at the extension step and identify the residue responsible for this selectivity. This residue acts as a kinetic switch, shunting repair toward long-patch BER upon correct dCMP incorporation, thus enhancing repair efficiency. Moreover, this switch also provides a potential mechanism to increase repair fidelity of MUTYH-initiated BER.

Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia.

Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.