Single-stranded DNA-binding Protein in Vitro Eliminates the Orientation-dependent Impediment to Polymerase Passage on CAG/CTG Repeats

Abstract

Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5-CAG and 5-CTG repeats that can be relieved by the binding of single-stranded DNAbinding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5-CAG is the template. We propose a "template-push" model in which the slow speed of DNA polymerase across the 5-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over. Expansion of simple trinucleotide repeats (TNRs)4 is the underlying genetic defect of a number of human neurodegenerative diseases. TNR-associated pathophysiology in human diseases strictly depends on the number of trinucleotide repeats (reviewed in Refs. 1-3). Although expansion can occur in germ cells (4) and in somatic cells with age (5, 6), emerging evidence suggests that rapid cell division in the early embryo can reduce the size of the repeats and modulate disease potential (7). TNR deletion during rapid cell division may serve as a natural defense mechanism for keeping the length of the repeats in check at critical times in development. Reducing the length of the repeat tract in disease genes is being explored as a therapeutic strategy. However, the mechanisms by which deletions rather than expansions are favored during replication remain poorly understood.DNA polymerase and strand slippage has been proposed as the primary mechanism for instability of trinucleotide repeats (1-3). During replication, the TNR units can misalign resulting in an extrahelical DNA loop that increases TNR length if it occurs on the daughter strand and decreases TNR length if it occurs on the template strand (1-3). In both prokaryotic and eukaryotic models, it is well established that TNR instability is sensitive to the length of the repeat (8 -12), to its structure forming potential (13-17), and to its proximity to the origin of replication (18). The factors that regulate the probability and frequency of deletion events, however, remain poorly understood. By a polymerase slippage model, a deletion implies that hairpin formation has occurred on the template strand. Theoretically, for an identical sequence located either on the template or daughter strand, slippage and hairpin formation can occur with equivalent frequen...

Hijacking of the mismatch repair system to cause CAG expansion and cell death in neurodegenerative disease

“…The impediment occurs only on the leading strand because SSB protein eliminates the impediment on both sequences [60]. This outcome indicates that the instability would be driven on the leading strand, consistent with the observation that instability is greater when 5'CTG is in the lagging strand.…”

“…If some aspect of the lagging strand configuration promotes slippage, there is no compelling explanation for why it should be limited to the template strand. Recent in vitro experiments have provided, however, at least one explanation [60].…”

“…In those experiments, both 5'CAG and 5'CTG repeats inhibits polymerase progression, but 5'-CAG is the most severe [60]. The impediment occurs only on the leading strand because SSB protein eliminates the impediment on both sequences [60].…”

“…5'-CTG or 5'CGG repeats have greater susceptibility to deletions when they serve as a template for lagging strand DNA synthesis. However, as the leading strand template, 5'-CTG or 5' CGG repeats are relatively stable [31,[49][50][51][52][53][54][60][61][62][63][64][65]. Thus, it is not obvious why slippage should be limited to only one orientation if the same sequence is being copied [66].…”

Mechanistic Studies of Hairpin to Duplex Conversion for Trinucleotide Repeat Sequences

“…Previous work using CAG and CTG sequences containing 10 repeats has shown that these sequences form stable hairpins; moreover, it is known that these complementary sequences can also form duplex (11)(12)(13). However, the mechanism by which the complementary hairpins interact to convert to duplex has not been established.…”