Kinetic Analyses of Single-Strand Break Repair by Human DNA Ligase III Isoforms Reveals Biochemical Differences from DNA Ligase I [Enzymology]

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 (LIG3α) and a germ line-specific splicing isoform (LIG3β) that differ in the C-terminal domain. Both isoforms are found in the nucleus and the mitochondria. Here, we determined the kinetics and thermodynamics of single-strand break ligation by LIG3α and LIG3β, 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 the BRCT domain specific to LIG3α 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 7 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 10 times weaker than that of LIG1, suggesting that Mg2+ availability regulates DNA ligation in vivo, as 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.

from # All Medicine by Alexandros G. Sfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2w4STuI
via IFTTT