Large-scale contractions of Friedreich’s ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability

Friedreich’s ataxia (FRDA) is a human hereditary disease caused by the presence of expanded (GAA)n repeats in the first intron of the FXN gene [V. Campuzano et al., Science 271, 1423–1427 (1996)]. In somatic tissues of FRDA patients, (GAA)n repeat tracts are highly unstable, with contractions more common than expansions [R. Sharma et al., Hum. Mol. Genet. 11, 2175–2187 (2002)]. Here we describe an experimental system to characterize GAA repeat contractions in yeast and to conduct a genetic analysis of this process. We found that large-scale contraction is a one-step process, resulting in a median loss of ∼60 triplet repeats. Our genetic analysis revealed that contractions occur during DNA replication, rather than by various DNA repair pathways. Repeats contract in the course of lagging-strand synthesis: The processivity subunit of DNA polymerase δ, Pol32, and the catalytic domain of Rev1, a translesion polymerase, act together in the same pathway to counteract contractions. Accumulation of single-stranded DNA (ssDNA) in the lagging-strand template greatly increases the probability that (GAA)n repeats contract, which in turn promotes repeat instability in rfa1, rad27, and dna2 mutants. Finally, by comparing contraction rates for homopurine-homopyrimidine repeats differing in their mirror symmetry, we found that contractions depend on a repeat’s triplex-forming ability. We propose that accumulation of ssDNA in the lagging-strand template fosters the formation of a triplex between the nascent and fold-back template strands of the repeat. Occasional jumps of DNA polymerase through this triplex hurdle, result in repeat contractions in the nascent lagging strand.

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Effects of Friedreich's ataxia GAA repeats on DNA replication in mammalian cells

Nucleic Acids Research ◽

10.1093/nar/gks021 ◽

2012 ◽

Vol 40 (9) ◽

pp. 3964-3974 ◽

Cited By ~ 27

Author(s):

Gurangad S. Chandok ◽

Mayank P. Patel ◽

Sergei M. Mirkin ◽

Maria M. Krasilnikova

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Gaa Repeats

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Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015699 ◽

2020 ◽

Vol 295 (47) ◽

pp. 15883-15891 ◽

Cited By ~ 1

Author(s):

Melanie A. Sparks ◽

Peter M. Burgers ◽

Roberto Galletto

Keyword(s):

Transcription Factors ◽

Dna Replication ◽

Dna Binding ◽

Dna Synthesis ◽

Dna Polymerase ◽

Strand Displacement ◽

Chromosomal Dna ◽

Dna Polymerase Δ ◽

Lagging Strand ◽

Stimulation Of

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.

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Faculty Opinions recommendation of Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1022005.252626 ◽

2004 ◽

Author(s):

Michael Lieber

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Dna Polymerase Delta ◽

Lagging Strand

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Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich’s Ataxia Cells

Cell Reports ◽

10.1016/j.celrep.2016.06.075 ◽

2016 ◽

Vol 16 (5) ◽

pp. 1218-1227 ◽

Cited By ~ 26

Author(s):

Jeannine Gerhardt ◽

Angela D. Bhalla ◽

Jill Sergesketter Butler ◽

James W. Puckett ◽

Peter B. Dervan ◽

...

Keyword(s):

Dna Replication ◽

Friedreich’S Ataxia ◽

Repeat Expansion ◽

Friedreich's Ataxia ◽

Replication Forks ◽

Gaa Repeats

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Prokaryotic DNA replication mechanisms

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1987.0068 ◽

1987 ◽

Vol 317 (1187) ◽

pp. 395-420 ◽

Cited By ~ 57

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Replication Fork ◽

Kinetic Studies ◽

Dna Helicase ◽

Gene 32 ◽

Dna Template ◽

Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

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Idling by DNA polymerase maintains a ligatable nick during lagging-strand DNA replication

Genes & Development ◽

10.1101/gad.1252304 ◽

2004 ◽

Vol 18 (22) ◽

pp. 2764-2773 ◽

Cited By ~ 154

Author(s):

P. Garg

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Lagging Strand

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Replication Stalling at Friedreich's Ataxia (GAA)n Repeats In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.24.6.2286-2295.2004 ◽

2004 ◽

Vol 24 (6) ◽

pp. 2286-2295 ◽

Cited By ~ 132

Author(s):

Maria M. Krasilnikova ◽

Sergei M. Mirkin

Keyword(s):

Dna Replication ◽

Length Polymorphism ◽

Direct Proof ◽

Friedreich’S Ataxia ◽

Repeat Expansion ◽

Friedreich's Ataxia ◽

Replication Fork Progression ◽

Threshold Length ◽

Replication Intermediates

ABSTRACT Friedreich's ataxia (GAA) n repeats of various lengths were cloned into a Saccharymyces cerevisiae plasmid, and their effects on DNA replication were analyzed using two-dimensional electrophoresis of replication intermediates. We found that premutation- and disease-size repeats stalled the replication fork progression in vivo, while normal-size repeats did not affect replication. Remarkably, the observed threshold repeat length for replication stalling in yeast (∼40 repeats) closely matched the threshold length for repeat expansion in humans. Further, replication stalling was strikingly orientation dependent, being pronounced only when the repeat's homopurine strand served as the lagging strand template. Finally, it appeared that length polymorphism of the (GAA) n · (TTC) n repeat in both expansions and contractions drastically increases in the repeat's orientation that is responsible for the replication stalling. These data represent the first direct proof of the effects of (GAA) n repeats on DNA replication in vivo. We believe that repeat-caused replication attenuation in vivo is due to triplex formation. The apparent link between the replication stalling and length polymorphism of the repeat points to a new model for the repeat expansion.

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Replication-independent instability of Friedreich’s ataxia GAA repeats during chronological aging

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013080118 ◽

2021 ◽

Vol 118 (5) ◽

pp. e2013080118

Author(s):

Alexander J. Neil ◽

Julia A. Hisey ◽

Ishtiaque Quasem ◽

Ryan J. McGinty ◽

Marcin Hitczenko ◽

...

Keyword(s):

Large Scale ◽

Disease Onset ◽

Experimental System ◽

Friedreich’S Ataxia ◽

Friedreich's Ataxia ◽

Hereditary Diseases ◽

Dna Microsatellites ◽

Repeat Instability ◽

Chronological Aging ◽

Somatic Instability

Nearly 50 hereditary diseases result from the inheritance of abnormally long repetitive DNA microsatellites. While it was originally believed that the size of inherited repeats is the key factor in disease development, it has become clear that somatic instability of these repeats throughout an individual’s lifetime strongly contributes to disease onset and progression. Importantly, somatic instability is commonly observed in terminally differentiated, postmitotic cells, such as neurons. To unravel the mechanisms of repeat instability in nondividing cells, we created an experimental system to analyze the mutability of Friedreich’s ataxia (GAA)n repeats during chronological aging of quiescent Saccharomyces cerevisiae. Unexpectedly, we found that the predominant repeat-mediated mutation in nondividing cells is large-scale deletions encompassing parts, or the entirety, of the repeat and adjacent regions. These deletions are caused by breakage at the repeat mediated by mismatch repair (MMR) complexes MutSβ and MutLα and DNA endonuclease Rad1, followed by end-resection by Exo1 and repair of the resulting double-strand breaks (DSBs) via nonhomologous end joining. We also observed repeat-mediated gene conversions as a result of DSB repair via ectopic homologous recombination during chronological aging. Repeat expansions accrue during chronological aging as well—particularly in the absence of MMR-induced DSBs. These expansions depend on the processivity of DNA polymerase δ while being counteracted by Exo1 and MutSβ, implicating nick repair. Altogether, these findings show that the mechanisms and types of (GAA)n repeat instability differ dramatically between dividing and nondividing cells, suggesting that distinct repeat-mediated mutations in terminally differentiated somatic cells might influence Friedreich’s ataxia pathogenesis.

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Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair

10.1101/2019.12.17.879544 ◽

2019 ◽

Author(s):

Natasha C Koussa ◽

Duncan J. Smith

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Genome Instability ◽

Dna Polymerase Delta ◽

Origin Firing ◽

A Genome ◽

Synthesis And Processing ◽

The Impact ◽

Lagging Strand

ABSTRACTDNA polymerase delta (Pol δ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol δ is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol δ has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol δ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol δ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol δ impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol δ depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol δ is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol δ is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol δ is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability.SIGNIFICANCE STATEMENTDNA replication in eukaryotes is carried out by the replisome – a multi-subunit complex comprising the enzymatic activities required to generate two intact daughter DNA strands. DNA polymerase delta (Pol δ) is a multi-functional replisome enzyme responsible for synthesis and processing of the lagging-strand. Mutations in Pol δ cause a variety of human diseases: for example, destabilizing mutations lead to immunodeficiency. We titrate the concentration of Pol δ in budding yeast – a simple model eukaryote with conserved DNA replication machinery. We characterize several replication defects associated with Pol δ scarcity. The defects we observe provide insight into how destabilizing Pol δ mutations lead to genome instability.

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