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Kinetic analyses of single-stranded break repair by human DNA ligase III isoforms reveal biochemical differences from DNA ligase I.

ABSTRACT: Humans have three genes encoding DNA ligases with conserved structural features and activities, but they also have notable differences. The LIG3 gene encodes a ubiquitous isoform in all tissues (LIG3?) and a germ line-specific splicing isoform (LIG3?) that differs in the C-terminal domain. Both isoforms are found in the nucleus and the mitochondria. Here, we determined the kinetics and thermodynamics of single-stranded break ligation by LIG3? and LIG3? and compared this framework to that of LIG1, the nuclear replicative ligase. The kinetic parameters of the LIG3 isoforms are nearly identical under all tested conditions, indicating that the BRCA1 C terminal (BRCT) domain specific to LIG3? does not alter ligation kinetics. Although LIG3 is only 22% identical to LIG1 across their conserved domains, the two enzymes had very similar maximal ligation rates. Comparison of the rate and equilibrium constants for LIG3 and LIG1 nevertheless revealed important differences. The LIG3 isoforms were seven times more efficient than LIG1 at ligating nicked DNA under optimal conditions, mainly because of their lower Km value for the DNA substrate. This could explain why LIG3 is less prone to abortive ligation than LIG1. Surprisingly, the affinity of LIG3 for Mg2+ was ten times weaker than that of LIG1, suggesting that Mg2+ availability regulates DNA ligation in vivo, because Mg2+ levels are higher in the mitochondria than in the nucleus. The biochemical differences between the LIG3 isoforms and LIG1 identified here will guide the understanding of both unique and overlapping biological roles of these critical enzymes.

SUBMITTER: McNally JR

PROVIDER: S-EPMC5612117 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Kinetic analyses of single-stranded break repair by human DNA ligase III isoforms reveal biochemical differences from DNA ligase I.

McNally Justin R JR O'Brien Patrick J PJ

The Journal of biological chemistry 20170727 38

Humans have three genes encoding DNA ligases with conserved structural features and activities, but they also have notable differences. The <i>LIG3</i> gene encodes a ubiquitous isoform in all tissues (LIG3α) and a germ line-specific splicing isoform (LIG3β) that differs in the C-terminal domain. Both isoforms are found in the nucleus and the mitochondria. Here, we determined the kinetics and thermodynamics of single-stranded break ligation by LIG3α and LIG3β and compared this framework to that ...[more]

PMID: 28751376

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Project description:During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

Dataset Information

Kinetic analyses of single-stranded break repair by human DNA ligase III isoforms reveal biochemical differences from DNA ligase I.

Publications

Kinetic analyses of single-stranded break repair by human DNA ligase III isoforms reveal biochemical differences from DNA ligase I.

Similar Datasets

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