Structural and functional analysis of split DNA ligases from T5-like bacteriophages

Abstract

T5-like bacteriophages, which infected Gram-negative bacteria, had persistent single-stranded nicks in one strand of their genomes at the consensus sequence 5’-GCGC. They also encoded an unusual NAD+-dependent heterodimeric DNAligase comprising A- and B-subunits. This project aimed to discover, using structural and biochemical methods, how the two ligase subunits functioned together and separately, and whether the split architecture was related to the nicks in the genome.

Solution structural analysis by small angle X-ray scattering (SAXS) showed the co-expressed split DNA ligase had an extended and flexible architecture in the absence of DNA, similar to the E. coli LigA (EcoLigA). X-ray crystallographic analysis of the PR1 (T5-like) DNA ligase in complex with a nicked dsDNA at stage two of the ligation process (PR1-DNA), revealed the A- and B-subunits encircled the DNA in the same manner as the EcoLigA. It also revealed the structure and role of the C-terminal BRCT domain in an NAD+-dependent DNA ligase for the first time. Interactions between the DNA ligase and DNA were not sequence-specific, consistent with the lack of preference for ligation of random sequences over 5’-GCGC sequences by in vitro biochemical assays.

In the phage genome, three promoters controlled the expression of the A- and B-subunits (one for the A-subunit, two for the B-subunit). Thus, there may be unequal amounts or different expression times of the subunits. An excess of the B-subunit decreased the ligation of DNA substrates with both random and 5’-GCGC sequences, in in vitro assays. Electrophoresis mobility shift assays revealed the B-subunit can independently bind to DNA, with a preference for the nicked DNA and the 5’-GCGC sequence. Models of the B-subunit in complex with a 26-mer nicked dsDNA, adapted from RoseTTAFold2NA and validated by SEC-SAXS, showed all the three domains interact with DNA, in contrast to the interactions seen in the PR1-DNA crystal structures. Analysis of DNA binding affinities by Microscale Thermophoresis using B-subunit mutants revealed key residues that recognise the nicked DNA and the 5’-GCGC sequence. These results supported the T5B-DNA model and suggested the BRCT domain recognises the phage genome nicks.

A molecular mechanism was proposed for the DNA ligation, whereby the B-subunit first contacted DNA through its BRCT domain recognising the nicked DNA, followed by a conformational change allowing the catalytic A-subunit to bind and ligate the nick. Excess B-subunit preferentially bound and protected the 5’-GCGC nicks, contributing to their persistence in the bacteriophage genome.