Nuclease Activity of Saccharomyces cerevisiae Mre11 Functions in Targeted Nucleotide Alteration

Abstract

Oligonucleotides can be used to direct site-specific changes in genomic DNA through a process in which mismatched base pairs in the oligonucleotide and the target DNA are created. The mechanism by which these complexes are developed and resolved is being studied by using Saccharomyces cerevisiae as a model system. Genetic analyses have revealed that in all likelihood the reaction occurs in two phases: DNA pairing and DNA repair. While the former phase involves strand assimilation, the latter phase likely involves an endonucleolytic processing step that leads to joint resolution. In this study, we established the importance of a functioning MRE11 gene in the overall reaction, as yeast strains deficient in MRE11 exhibited severely reduced activity. The activity could be rescued by complementation with wild-type MRE11 genes but not with MRE11 alleles lacking the nuclease function. Taken together, the data suggest that Mre11 provides nuclease activity for targeted nucleotide exchange, a process that could be used to reengineer yeast genes.Genetic reengineering in lower eukaryotes holds promise for the creation of host strains that can produce useful metabolites or express desirable phenotypes. The majority of efforts in yeasts have focused on the development of knock-out strategies, where the aim is to completely disable the gene but in many cases the most desirable outcome is a (subtle) modulation of gene function. Thus, new approaches that are simple and straightforward and do not require multiple cloning steps must be developed to meet this goal. A direct approach centers around alteration of single bases at specific sites in chromosomal genes without the need for integration or extensive genetic rearrangement.Single-stranded DNA molecules can be used to direct the alteration or exchange of specific nucleotides in the genome of Saccharomyces cerevisiae. Pioneering work by Moerschell and colleagues (16) demonstrated the feasibility of base repair in the CYC1 gene, a gene encoding cytochrome c isoform 1. A variety of point and frameshift mutations within CYC1 were reversed by the transformation of single strands of DNA in approximately 0.0003 to 0.002% of the cells (28). The mechanism of conversion, however, was not fully elucidated in these studies, although the reaction was found to be dose dependent and exhibited an interesting strand bias. In a similar line of experiments, we have begun to examine the potential for using modified single-stranded DNA oligonucleotides (MSOs) to repair point and frameshift mutations in the yeast chromosome, and our long-term goal is to apply the technique to mammalian cells (22; see reference 2 for a review). To this end, we focused on examining the mechanism by which MSOs direct nucleotide alteration in yeast cells.

Real-Time PCR Quantification and Diversity Analysis of the Functional Genes aprA and dsrA of Sulfate-Reducing Prokaryotes in Marine Sediments of the Peru Continental Margin and the Black Sea

“…Members of the genera Desulfococcus and Desulfobacterium belong to the family of Desulfobacteraceae that are known to be able to oxidize a great variety of different electron donors completely to CO 2 . Thus, they successfully inhabit anoxic marine environments such as Black Sea and Peru margin sediments (Ravenschlag et al, 2000; Liu et al, 2003; Mußmann et al, 2005; Kondo et al, 2007; Leloup et al, 2007, 2009) or the anoxic water column of the Black Sea (Vetriani et al, 2003; Neretin et al, 2007) and other marine habitats (Kondo et al, 2007). Besides dsrA sequences affiliated to Desulfobacteraceae , also numerous aprA sequences belonging to this family were detected in samples from the Black Sea and Peru margin sediments indicating that bacteria of this community play an important role in sulfate reduction in these sediments.…”

Gene therapy progress and prospects: targeted gene repair

“…Earlier studies examined the effect of single-stranded DNA molecules on mammalian cells, and found that the cell responds to the presence of free ends by arresting cell growth, consistent with this interpretation of the gene repair reaction. 53 Thus, the cell may have to pay a price for achieving high levels of correction and this could explain why the technique has not been as reproducible as predicted from the early reports of success. It may also explain the low or variable levels of correction often obtained in different systems in which corrected cells may have survived to varying degrees prior to analyses.…”

Increased efficiency of oligonucleotide-mediated gene repair through slowing replication fork progression

“…Analogous to the situation for RDOs, a two-step mechanism has been proposed, which involves the following steps: (i) DNA pairing͞annealing, followed by (ii) DNA repair͞recombination (18)(19)(20). First, the SSO (or the DNA component of the RDO) anneals with its complementary strand in the double-stranded DNA to form a D-loop structure, in a process mediated by cellular protein factors (13,(21)(22)(23). The formation of the D-loop, containing a centrally located mismatched base pair (in the case of a point mutation), results in a structural perturbation that most likely stimulates the endogenous protein machinery to initiate the second step in the repair process: site-specific modification of the chromosomal sequence.…”