Transcription-associated mutagenesis

Abstract

Transcription requires unwinding complementary DNA strands, generating torsional stress, and sensitizing the exposed single strands to chemical reactions and endogenous damaging agents. In addition, transcription can occur concomitantly with the other major DNA metabolic processes (replication, repair, and recombination), creating opportunities for either cooperation or conflict. Genetic modifications associated with transcription are a global issue in the small genomes of microorganisms in which noncoding sequences are rare. Transcription likewise becomes significant when one considers that most of the human genome is transcriptionally active. In this review, we focus specifically on the mutagenic consequences of transcription. Mechanisms of transcription-associated mutagenesis in microorganisms are discussed, as is the role of transcription in somatic instability of the vertebrate immune system.

Keywords: DNA damage; class-switch recombination; cytosine deamination; somatic hypermutation; topoisomerase.

Figure 1

Effects of transcription on the DNA template. The transcription bubble and a trailing R-loop are indicated as small and large rectangles, respectively. Circles indicate normal intertwining of DNA strands; compressed or extended ovals correspond to over- or underwound strands, respectively, and the regions of associated positive (+) or negative (−) supercoils are indicated. RNAP is depicted as a blue oval, and the blue arrow indicates its direction of movement on the DNA template. DNA and RNA strands are black and red, respectively; yellow triangles indicate damage to ssDNA.

Figure 2

Conflicts between the replication and transcription machineries. Movement of the replisome and RNAP in the same or opposite direction can cause (a) codirectional or (b) head-on conflicts, respectively. Red and black lines represent RNA and DNA, respectively; dashed lines depict newly synthesized DNA; blue ovals represent RNAP. Yellow and blue arrows indicate the direction of the replication fork and RNAP movement, respectively.

Figure 3

Inferring strand specificity from mutation patterns associated with cytosine deamination. Yellow and pink boxes indicate consequences of cytosine deamination on the nontranscribed strand (NTS) and transcribed strand (TS), respectively.

Figure 4

Mechanisms of Top1 mutagenesis in transcriptionally active DNA. Two distinct mechanisms of Top1-dependent mutagenesis are shown, with a hypothetical dinucleotide repeat highlighted in gray. When Top1 incision occurs, the active-site tyrosine forms a covalent linkage to the 3′-PO₄ on one side of the DNA nick, leaving a 5′-OH on the other side. (a) Top1 becomes trapped as a stabilized cleavage complex (step i), and its removal by unknown proteins generates a 2-nt gap within the 2-bp tandem repeat (step ii). Realignment of the DNA strands converts the gap to a nick (step iii), which facilitates ligation and produces the mutation intermediate (step iv). (top) Replication of the newly ligated strand results in a permanent, 2-bp deletion (step v); replication of the other strand results is of no genetic consequence. (b) Top1 incises at the position of an rNMP (red R). The 2′-OH of ribose attacks the phosphotyrosyl bond, releasing Top1 and generating a 2′,3′-cyclic phosphate (red triangle; step i). A second incision by Top1 upstream of the nick (step ii) releases the intervening oligonucleotide and transiently traps the covalent enzyme-DNA intermediate (step iii). Realignment of the two DNA strands by the repeat sequence correctly orients the Top1-DNA complex and the 5′-OH, enabling efficient Top1-mediated rejoining of the ends (step iv). Replication of the top strand fixes the 2-bp deletion (step v).

Figure 5

A model for somatic hypermutation (SHM) and class-switch recombination (CSR) during antibody maturation. Different sequence elements are shown as rectangles or ovals of different colors. The direction of transcription is indicated by a rightward arrow. AID is recruited at a proximal pause site for RNAP II. Following release from the pause, AID travels with RNAP II as it transcribes DNA and converts cytosine to uracil on each DNA strand. Uracil is excised by uracil N-glycosylase (UNG) and processed by error-prone base-excision repair (BER) or by mismatch repair (MMR) to introduce point mutations, which are indicated by asterisks. To initiate CSR, double-strand breaks are generated by AP endonuclease (64a) incision at UNG-generated apurinic/apyrimidinic AP sites. Broken ends are ligated by the nonhomologous end-joining (NHEJ) pathway, and the intervening DNA is released as a switch circle. Abbreviations: C, constant segment (only the μ, ε, α regions are shown); S, switch region preceding each C segment; V(D)J, variable segment.