Functional analysis of subcellular localization and protein-protein interaction sequences in the essential DNA ligase I protein of fission yeast

Abstract

DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus. In the budding yeast Saccharomyces cerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair. In this report, dual nuclear-mitochondrial localization is demonstrated to be a property of the essential Lig I enzyme Cdc17 from the distantly related fission yeast Schizosaccharomyces pombe. Expression of nuclear and mitochondrial forms of Cdc17 from separate genes shows that, whereas expression of either protein alone is insufficient to restore viability to cells lacking endogenous Cdc17, co-expression restores full viability. In the nucleus, Lig I interacts with the sliding clamp proliferating cell nuclear antigen (PCNA) via a conserved PCNA interacting sequence motif known as a PIP box. Deletion of the PIP motif from the N-terminus of the nuclear form of Cdc17 fails to abolish Cdc17 function, indicating that PCNA binding by Cdc17 is not an absolute requirement for completion of S-phase.

Adaptive expression responses in the Pol-γ null strain of S. pombe depleted of mitochondrial genome

“…For this reason, we constructed a strain bearing a sole copy of pog1 + -GFP that exhibited no abnormal growth phenotypes, suggesting that C-terminus tagging of Pol-γ does not affect its biological function. The GFP-tagged Pol-γ showed the subcellular localization resembling to that of mitochondria previously reported [ 16 , 17 ] (Figure 2B ).…”

Genetic Instability Induced by Overexpression of DNA Ligase I in Budding Yeast

“…In both TNR orientations in wild-type cells, overexpression of either of the PCNA interaction mutants gave the same expansion (yJS1 and yJS2) and contraction rates (yJS3 and yJS4) as control cells (four strains transformed with pRS415; Table 2). We were not surprised that both CDC9 constructs were able to complement the temperature sensitivity of LP2915-8D when overexpressed ( Figure 1A) since a PCNA-DNA ligase I interaction is not required for viability in yeast (Sriskanda et al 1999;Martin and Macneill 2004). Increasing the level of DNA ligase I enzymatic activity, by overexpressing mutant versions of GST-CDC9 that lack PCNA binding, does not enhance expansion rates.…”

An evolutionarily conserved translation initiation mechanism regulates nuclear or mitochondrial targeting of DNA ligase 1 in Arabidopsis thaliana

“…Therefore the nucleus, mitochondria and chloroplast must all import DNA ligase enzyme protein that is translated on cytoplasmic ribosomes using mRNA transcripts expressed from nuclear genes. Intriguingly, rather than having distinct genes within their nuclear genomes to code for the different multi‐compartmentalized isoforms of DNA ligase proteins, eukaryotic species from yeast (Martin and MacNeill, 2004; Willer et al. , 1999) to humans (Lakshmipathy and Campbell, 1999) appear to utilize an evolutionarily conserved mechanism that relies upon choice of in‐frame translation initiation start codons within the ORF of a ligase mRNA transcript to regulate the synthesis of the appropriate DNA ligase isoform destined for the nucleus or mitochondria (DNA ligase I isoforms in yeasts and plants and DNA ligase III α isoforms in animals).…”

“…Mitochondria and chloroplast genomes do not encode a DNA ligase, and this activity must be imported from the cytosol into both organelles as well as into the nucleus. In humans (Lakshmipathy and Campbell, 1999) and yeast (Martin and MacNeill, 2004; Willer et al. , 1999), a single ligase transcript (arising from DNA ligase III and I genes, respectively) can be translated from separate in‐frame initiation codons in the ligase ORF to produce topologically distinct isoforms of DNA ligase protein localized to more than one cell compartment.…”