Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.

Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.

Expansions, contractions, and fragility of the spinocerebellar ataxia type 10 pentanucleotide repeat in yeast.

Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT)(n) repeat within an intron of the human ATXN10 gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra- or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT)(n) repeat in a yeast experimental system, where its expansions led to inactivation of the URA3 reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional TOF1 gene precludes, whereas a functional RAD5 gene promotes, expansions of the (ATTCT)(n) repeat. We hypothesize that repeat expansions could occur upon fortuitous template switching during DNA replication. The rate of repeat contractions was elevated in the Tof1 knockout strain, but it was not affected by the RAD5 gene. Supporting the notion of replication irregularities, we found that (ATTCT)(n) repeats also cause length-dependent chromosomal fragility in yeast. Repeat-mediated fragility was also affected by the Tof1 and Rad5 proteins, being reduced in their absence.

Using Implementation Frameworks to Provide Proactive Physical Therapy for People With Parkinson Disease: Case Report.

BACKGROUND AND PURPOSE: European clinical practice guidelines recommend physical therapy for people with Parkinson disease (PD) soon after diagnosis to provide education, physical activity advice, and individualized interventions when needed. However, therapy is frequently not used until after gait and balance problems occur. The purpose of this administrative case study is to present the application of a proactive physical therapy (PAPT) approach at 1 rehabilitation center using implementation frameworks to support the (1) implementation process, (2) determinants of implementation success, and (3) implementation evaluation. CASE DESCRIPTION: The PAPT program targeted people with PD before the onset of significant mobility dysfunction. It was initiated in 1 outpatient neurological rehabilitation center. The program used shared decision-making to promote long-term maintenance of independent exercise. The Knowledge-to-Action Framework was used by champions to plan implementation processes. Implementation barriers were addressed using the Consolidated Framework for Implementation Research. The program was evaluated using the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) framework with mixed methods. OUTCOMES: In the program's first year, 38 people were referred, 28 were evaluated, and 20 participated in the 6-month program evaluation. Following PAPT, the number of participants reporting regular participation in aerobic, strengthening, and flexibility exercise approximately doubled, while those engaging in balance activities increased from 1 to 8. They reported a median of 140 minutes of aerobic exercise per week. Implementation barriers included location, insurance coverage, and difficulty scheduling long-term follow-up visits. Participants reported physical and emotional benefits of the program. DISCUSSION: Implementation frameworks assisted with the implementation and evaluation of a PAPT delivery model that helped people with PD to increase and maintain independent exercise participation.