MD simulations of EXOG and its mutants with ssDNA and ssRNA

AbstractSingle-stranded DNA (ssDNA) is an important intermediate generated during various cellular DNA transactions, primarily during long-patch base excision repair. When displaced by DNA polymerase during strand displacement DNA synthesis, ssDNA forms 5′ overhangs (flaps) that are either cleaved by DNA nucleases or protected from degradation upon binding of single-stranded DNA binding proteins (SSB). There are several nucleases implicated in ssDNA flaps removal in human mitochondria, namely endonucleases FEN1 and DNA2 as well as exonuclease MGME1. In this study, we show that another mitochondrial nuclease, EXOG, cleaves DNA flaps in both free and SSB-protected forms. We established that the presence of the Wing domain in EXOG structure provides additional binding site for ssDNA and 5′ flaps irrespective of monovalent salt concentration. Importantly, DNA flap cleavage by EXOG is compatible with the activity of other mitochondrial enzymes involved in DNA replication/repair, e.g., mtSSB, Pol γ and Lig III, as we were able to reconstitute a multistep reaction of DNA synthesis, flap removal and nick ligation. Our findings highlight the versatile role of EXOG in maintaining mitochondrial DNA integrity, adding single-stranded DNA flap removal to the repertoire of its DNA processing activities.MethodsThe dataset is collected through molecular dynamics simulations using GROMACS.