Abstract
Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2–7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.
Highlights
Expanded CAG repeats are responsible for several inherited neurodegenerative diseases including Huntington’s disease (HD), myotonic dystrophy type 1 (DM1) and several types of spinocerebellar ataxia (SCA) [1,2]
Trinucleotide repeat expansions are the cause of several muscular- and neuro-degenerative diseases, and further expansions during intergenerational inheritance often leads to an earlier age-of-onset in the offspring
We discovered that a restarted replication fork traversing a CAG repeat tract is highly error prone and repeat expansions and contractions are more prevalent in this case compared to normal replication
Summary
Expanded CAG repeats are responsible for several inherited neurodegenerative diseases including Huntington’s disease (HD), myotonic dystrophy type 1 (DM1) and several types of spinocerebellar ataxia (SCA) [1,2]. Hairpin formation during replication or repair can lead to repeat length changes referred to as repeat instability, including expansions and contractions [4,5]. CAG repeat expansions have been shown to occur both in germ cells and somatic cells [6,7]. Instability during non-replicating somatic cells likely occurs during gap repair and is dependent on mismatch repair proteins, primarily Msh2-Msh and Mlh1-Mlh3 [8]. Repeat instability during replication may be especially relevant to intergenerational repeat expansions; for example, HD expansions can occur in premeiotic replicating testicular germ cells [9]
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