Understanding DNA Repair in Hyperthermophilic Archaea: Persistent Gaps and Other Reasons to Focus on the Fork

Abstract

Although hyperthermophilic archaea arguably have a great need for efficient DNA repair, they lack members of several DNA repair protein families broadly conserved among bacteria and eukaryotes. Conversely, the putative DNA repair genes that do occur in these archaea often do not generate the expected phenotype when deleted. The prospect that hyperthermophilic archaea have some unique strategies for coping with DNA damage and replication errors has intellectual and technological appeal, but resolving this question will require alternative coping mechanisms to be proposed and tested experimentally. This review evaluates a combination of four enigmatic properties that distinguishes the hyperthermophilic archaea from all other organisms: DNA polymerase stalling at dU, apparent lack of conventional NER, lack of MutSL homologs, and apparent essentiality of homologous recombination proteins. Hypothetical damage-coping strategies that could explain this set of properties may provide new starting points for efforts to define how archaea differ from conventional models of DNA repair and replication fidelity.

Figure 1

Possible fates of a replicative polymerase stalled at template dU. (a) Archaeal polymerases stall with the nascent 3′ end 4 nt ahead of the template dU [23]. (b) If the replication fork responds with immediate fork reversal, the dU is restored to its original context, and BER is required for resumption of fork progress. (c) If tight coupling is not preserved, the Mcm helicase and nonstalled polymerase (not drawn) would continue, creating ssDNA region on the stalled template vulnerable to structure- or single-strand-specific nucleases. Arrowheads on DNA strands represent 3′ ends.

Figure 2

Fork breakage “collapse” in response to replication-blocking lesions. Adducts or other large, helix-distorting lesions (symbolized by X) block a polymerase molecule, inducing a certain degree of uncoupling and ssDNA exposure at the base of the fork. Cleavage by an appropriate single-strand-specific or structure-specific endonuclease can, for each case, generate dsDNA end that retains the lesion. The top strand in each DNA duplex is oriented with the 5′ end on left; free 3′ ends are represented by arrowheads, whereas broken lines indicate the rest of the chromosome. Carets (∧) locate the single cut that would be made by the indicated nuclease. For the cleavage preferences of archaeal Hef, Xpf, and Fen1/Xpg, see [36, 65, 66].

Figure 3

Patchy recombination in Sulfolobus. Cells transformed with linear DNAs marked at multiple sites (or mated with multiply marked donor cells) produce recombinants that indicate erratic, localized strand loss from heteroduplex intermediates [48, 67]. (a) Markers are acquired from ssDNA as multiple short tracts, some of which consist of a single marker. (b) A preformed heteroduplex, containing two distinct donor alleles at each marked position, generates similar short patches representing all three possible alleles. Semicircular symbols depict “silent” genetic markers (synonymous mutations), and the colors (black, white, and gray) depict different alleles. Different colors opposite to each other indicate a mismatch that is eventually resolved by strand removal and resynthesis.

Figure 4

Repositioning of replication errors by fork reversal. If a replication fork reverses shortly after making a replication error, the resulting mismatch would be localized to the short (extruded) arm. In principle, this could provide a basis for removing replication errors without involving a conventional (MutSL-dependent) MMR system. The white semicircle represents a polymerase error; heavy lines depict parental (template) strands.

Figure 5

Regeneration of broken replication forks by HR functions. (a) The products of archaeal replication fork breakage proposed in Figure 2 are expected to retain various lesions, gaps, and overhangs; possible structures are illustrated to the left of each bracket. These various structures must be converted to a “clean” 3′ extension on the ds end and an intact continuous duplex with which it can recombine. (b) Proteins of double-strand-break repair (homologous recombination) promote subsequent reassembly of the replication fork. The steps depicted here are those commonly proposed for bacteria and eukaryotes [37, 38, 52]. As in previous figures, broken lines indicate the remainder of the chromosome, and arrowheads mark 3′ ends.