Structures of LIG1 uncover the mechanism of sugar discrimination against a ribonucleotide at 3’- and 5’-end of the nick DNA

Abstract

Human DNA ligase I (LIG1) is the main replicative ligase that seals Okazaki fragments and finalizes DNA repair pathways by joining canonical 3’-OH and 5’-P ends of the nick DNA in a three-step ligation reaction. Ribonucleotides can be misincorporated by DNA polymerases resulting in a nick with 3’-ribonucleotide while RNase H2 mediated cleavage leaves a nick harboring 5’-ribonucleotide during ribonucleotide excision repair. However, how LIG1 surveils DNA ends with a “wrong” sugar at atomic resolution is unknown. Here, we determine X-ray structures of LIG1/nick DNA complexes with 3’- or 5’-single ribonucleotide during different stages of the ligation reaction. Our LIG1/5’-rG:C structure reveals a global conformational change, which discriminates against 5’-RNA/DNA junctions at the initial step when the ligase-AMP intermediate is formed. Furthermore, we capture LIG1/3’-RNA-DNA heteroduplexes that are tolerated at the active site where AMP is transferred to nick DNA (step 2) and final phosphodiester bond formation occurs (step 3). Finally, we demonstrate the mutagenic and defective ligation of the nick DNA with 3’- and 5’-ribonucleotide, respectively,in vitro. Together, these results uncover how LIG1 encounters ribonucleotides embedded into genome during nuclear replication and the last step of DNA repair pathways to maintain genome integrity.