Heteroduplex Formation, Mismatch Resolution, and Genetic Sectoring During Homologous Recombination in the Hyperthermophilic Archaeon Sulfolobus Acidocaldarius

Dominic Mao,Dennis W Grogan

doi:10.3389/fmicb.2012.00192

Abstract

Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR) remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldarius pyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr+ clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells, or to conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which regions of heteroduplex DNA formed and then segregated after partial resolution of inter-strand differences. Surprisingly, sectoring was also frequent in cells transformed with single-stranded DNAs. Oligonucleotides produced more sectored transformants when electroporated as single strands than as a duplex, although all forms of donor DNA (positive-strand, negative-strand, and duplex) produced a diversity of genotypes, despite the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells. Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.

Highlights

All cellular organisms can transfer strands between two DNA molecules of nearly identical sequences in an active process termed homologous recombination
STRAINS AND GROWTH CONDITIONS The Sulfolobus strain used for most experiments was S. acidocaldarius strain MR31, which was derived from his-2 mutant DG55 and has an internal deletion of the pyrE gene removing nt 154–171 (Reilly and Grogan, 2001)
GENETIC EVIDENCE OF HETERODUPLEX SEGREGATION To investigate whether heteroduplex DNA formed by homologous recombination (HR) can be detected in Sulfolobus spp., we electroporated strain MR31 with a pyrE DNA marked by a set of phenotypically silent mutations (Grogan and Rockwood, 2010)

Summary

Introduction

All cellular organisms can transfer strands between two DNA molecules of nearly identical sequences in an active process termed homologous (or generalized) recombination. Bacteria and eukaryotic cells use distinct sub-families of ssDNA-binding proteins (the RecA and Rad families, respectively) to promote strand exchange, and differ further with respect to the helicases, nucleases, and accessory proteins that interact to catalyze and regulate the steps before and after this exchange (Kowalczykowski et al, 1994; Aylon and Kupiec, 2004; Krogh and Symington, 2004) Consistent with their extremely early divergence from the bacterial and eukaryotic lineages, archaea encode a third, structurally distinct sub-family of strand-exchange proteins called the RadA family (Sandler et al, 1999). Archaea resemble bacteria with respect to certain features of cell and genome structure, archaeal RadA proteins most closely resemble the Rad and Dmc proteins of eukaryotes (Sandler et al, 1999)

Methods

Results

Conclusion