Folded DNA in action: hairpin formation and biological functions in prokaryotes

Abstract

Structured forms of DNA with intrastrand pairing are generated in several cellular processes and are involved in biological functions. These structures may arise on single-stranded DNA (ssDNA) produced during replication, bacterial conjugation, natural transformation, or viral infections. Furthermore, negatively supercoiled DNA can extrude inverted repeats as hairpins in structures called cruciforms. Whether they are on ssDNA or as cruciforms, hairpins can modify the access of proteins to DNA, and in some cases, they can be directly recognized by proteins. Folded DNAs have been found to play an important role in replication, transcription regulation, and recognition of the origins of transfer in conjugative elements. More recently, they were shown to be used as recombination sites. Many of these functions are found on mobile genetic elements likely to be single stranded, including viruses, plasmids, transposons, and integrons, thus giving some clues as to the manner in which they might have evolved. We review here, with special focus on prokaryotes, the functions in which DNA secondary structures play a role and the cellular processes giving rise to them. Finally, we attempt to shed light on the selective pressures leading to the acquisition of functions for DNA secondary structures.

FIG. 1.

Hairpin formation during replication. Hairpins can fold on the ssDNA formed by the discontinuous replication of the lagging strand or on ssDNA gaps remaining after lesion bypass.

FIG. 2.

Mechanisms of cruciform extrusion. In the C-type pathway, a substantial region of dsDNA is denatured, allowing the folding of the whole hairpins on both strands in one step. In the S-type pathway, a small region is denatured (∼10 bp), allowing the folding of a small hairpin that can then be elongated through branch migration.

FIG. 3.

Genetic instability of inverted repeats. (A) Formation of a hairpin on the template strand can lead to the deletion of the inverted repeat. (B) Hairpins can be cleaved by SbcCD, leading to double-strand breaks that can then be repaired through homologous recombination. (C) Imperfect inverted repeats can mutate toward perfection through a template switch mechanism where the first repeat becomes the template for the second repeat.

FIG. 4.

Priming of replication on ssDNA hairpins. In G4-type priming, hairpins structure the region, directing the binding of SSB and allowing access to the dnaG primase. In φX174-type priming, an ssDNA hairpin forms a Y fork recognized by PriA, which directs the formation of a primosome. In filamentous-phage-type priming, a hairpin mimicking a promoter is recognized by the RNA polymerase (RNAP), which synthesizes an RNA primer for replication.

FIG. 5.

Rolling-circle replication. (A) The Rep protein binds a hairpin formed by double-stranded origin (dso) and extruded from dbDNA as a cruciform. Rep nicks DNA and covalently binds the 5′ end, leaving a 3′ end for replication to proceed. The leading strand is replicated while the lagging strand is extruded and remains single stranded until the single-stranded origin (sso) is reached. The RNAP binds the sso hairpin and synthesizes an RNA primer for replication. (B) The pT181 dso in cruciform conformation. (C) The pT181 sso as folded by use of mFOLD software.

FIG. 6.

N4 virion hairpin promoters. Shown are the three promoters of N4 controlling the expression of the early genes as cruciform structures.

FIG. 7.

The V. cholerae chromosome I dif site and the CTX phage hairpin. The CTX attP region folds into a forked hairpin mimicking V. cholerae dif1. This enables the CTX phage to use the host XerCD recombinase to catalyze its integration into the chromosome.

FIG. 8.

Organization of IS608 and overall transposition pathway. (A) Organization. Shown are tnpA and tnpB open reading frames (light and dark arrows, respectively) and the left end (LE) and right end (RE) (red and blue boxes, respectively).(B) Sequence of the LE and RE. Sequence and secondary structures, IP_L and IP_R, at the LE and RE of IS608 are shown. Left and right tetranucleotide cleavage sites (C_L and C_R, respectively) are boxed in black (C_L) and underlined in blue (C_R). They are recognized by the B_L and B_R tetranucleotide boxes, respectively, through folding and unconventional base pairing. Also shown is the position of cleavage and of the formation of the 5′ phosphotyrosine TnpA-DNA intermediate (vertical arrows). (C) Transposition pathway. (i) Schematized IS608 with IP_L and IP_R and left (TTAC) (C_L) and right (TCAA) (C_R) cleavage sites. (ii) Formation of a single-strand transposon circle intermediate with abutted left and right ends. The transposon junction (TCAA) and donor joint (TTAC) are shown. (iii) Pairing with the target (TTAC) and cleavage (vertical arrows). (iv) Inserted transposon with new left and right flanks (dotted black lines). (Reprinted from reference with permission of the publisher.)

FIG. 9.

Recombination between an attC site hairpin of an integron cassette and a double-stranded attI site. The first recombination steps (A to C) between the folded attC site and the dsDNA attI site are identical to classical recombination steps catalyzed by other tyrosine recombinases. (B) Four integrase monomers bind to the core sites (with the proper strand of the attC site being recognized through specific binding with the extrahelical G). (C) Binding to structural determinants makes the pink monomers inactive, leaving the green monomers the possibility to realize the first strand exchange. The pseudo-Holliday junction formed cannot be resolved by a second strand exchange, as occurs with classical tyrosine recombinases. (D) The current model is that replication is involved to solve the junction in a process that remains to be understood.

FIG. 10.

ssDNA, at the crossroads of horizontal gene transfer, the SOS response, and genetic rearrangements. (1) Conjugation, transformation, phage infection, and environmental stress lead to the production of ssDNA in the cell. (2) The RecA proteins bind ssDNA and trigger the self-cleavage of LexA (brown circles). (3) The SOS regulon is derepressed, recombinases are expressed (orange triangles), and DNA coiling is modified. (4) Increased supercoiling leads to cruciform formation. (5) Induction of IS transposition and integron recombination. (6) ICE conjugation, lysogenic phages, and natural competence are induced.