Cooperative interaction of CST and RECQ4 resolves G-quadruplexes and maintains telomere stability.

Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that interacts with the replisome to aid in stalled fork rescue. We previously found that CST promotes telomere replication to maintain genomic integrity via G-quadruplex (G4) resolution. However, the detailed mechanism by which CST resolves G4s in vivo and whether additional factors are involved remains unclear. Here, we identify RECQ4 as a novel CST-interacting partner and show that RECQ4 can unwind G4 structures in vitro using a FRET assay. Moreover, G4s accumulate at the telomere after RECQ4 depletion, resulting in telomere dysfunction, including the formation of MTSs, SFEs, and TIFs, suggesting that RECQ4 is crucial for telomere integrity. Furthermore, CST is also required for RECQ4 telomere or chromatin localization in response to G4 stabilizers. RECQ4 is involved in preserving genomic stability by CST and RECQ4 disruption impairs restart of replication forks stalled by G4s. Overall, our findings highlight the essential roles of CST and RECQ4 in resolving G-rich regions, where they collaborate to resolve G4-induced replication deficiencies and maintain genomic homeostasis.

Checkpoint functions of RecQ helicases at perturbed DNA replication fork.

DNA replication checkpoint is a cell signaling pathway that is activated in response to perturbed replication. Although it is crucial for maintaining genomic integrity and cell survival, the exact mechanism of the checkpoint signaling remains to be understood. Emerging evidence has shown that RecQ helicases, a large family of helicases that are conserved from bacteria to yeasts and humans, contribute to the replication checkpoint as sensors, adaptors, or regulation targets. Here, we highlight the multiple functions of RecQ helicases in the replication checkpoint in four model organisms and present additional evidence that fission yeast RecQ helicase Rqh1 may participate in the replication checkpoint as a sensor.

Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis.

During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100-200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers. To bias DSB repair toward crossovers, we simultaneously increased dosage of the procrossover E3 ligase gene HEI10 and introduced mutations in the anticrossovers helicase genes RECQ4A and RECQ4B As HEI10 and recq4a recq4b increase interfering and noninterfering crossover pathways, respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases distally towards the subtelomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid.

Crossover formation during meiosis in plants is required for proper chromosome segregation and is essential for crop breeding as it allows an (optimal) combination of traits by mixing parental alleles on each chromosome. Crossover formation commences with the production of a large number of DNA double-strand breaks, of which only a few result in crossovers. A small number of genes, which drive the resolution of DNA crossover intermediate structures towards non-crossovers, have been identified in Arabidopisis thaliana. In order to explore the potential of modification of these genes in interspecific hybrids between crops and their wild relatives towards increased production of crossovers, we have used CRISPR/Cas9-mutagenesis in an interspecific tomato hybrid to knockout RecQ4. A biallelic recq4 mutant was obtained in the F1 hybrid of Solanum lycopersicum and S. pimpinellifolium. Compared with the wild-type F1 hybrid, the F1 recq4 mutant was shown to have a significant increase in crossovers: a 1.53-fold increase when directly observing ring bivalents in male meiocytes microscopically and a 1.8-fold extension of the genetic map when measured by analysing SNP markers in the progeny (F2) plants. This is one of the first demonstrations of increasing crossover frequency in interspecific hybrids by manipulating genes in crossover intermediate resolution pathways and the first to do so by directed mutagenesis. SIGNIFICANCE STATEMENT: Increasing crossover frequency during meiosis can speed up or simplify crop breeding that relies on meiotic crossovers to introduce favourable alleles controlling important traits from wild relatives into crops. Here we show for the first time that knocking out an inhibitor of crossovers in an interspecific hybrid between tomato and its relative wild species using CRISPR/Cas9-mutagenesis results in increased recombination between the two genomes.

Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM.

Meiotic crossovers (COs) have two important roles, shuffling genetic information and ensuring proper chromosome segregation. Despite their importance and a large excess of precursors (i.e., DNA double-strand breaks, DSBs), the number of COs is tightly regulated, typically one to three per chromosome pair. The mechanisms ensuring that most DSBs are repaired as non-COs and the evolutionary forces imposing this constraint are poorly understood. Here we identified Topoisomerase3α (TOP3α) and the RECQ4 helicases--the Arabidopsis slow growth suppressor 1 (Sgs1)/Bloom syndrome protein (BLM) homologs--as major barriers to meiotic CO formation. First, the characterization of a specific TOP3α mutant allele revealed that, in addition to its role in DNA repair, this topoisomerase antagonizes CO formation. Further, we found that RECQ4A and RECQ4B constitute the strongest meiotic anti-CO activity identified to date, their concomitant depletion leading to a sixfold increase in CO frequency. In both top3α and recq4ab mutants, DSB number is unaffected, and extra COs arise from a normally minor pathway. Finally, both TOP3α and RECQ4A/B act independently of the previously identified anti-CO Fanconi anemia of complementation group M (FANCM) helicase. This finding shows that several parallel pathways actively limit CO formation and suggests that the RECQA/B and FANCM helicases prevent COs by processing different substrates. Despite a ninefold increase in CO frequency, chromosome segregation was unaffected. This finding supports the idea that CO number is restricted not because of mechanical constraints but likely because of the long-term costs of recombination. Furthermore, this work demonstrates how manipulating a few genes holds great promise for increasing recombination frequency in plant-breeding programs.

Saccharomyces cerevisiae Hrq1 helicase activity is affected by the sequence but not the length of single-stranded DNA.

Mutations in the human RecQ4 DNA helicase are associated with three different diseases characterized by genomic instability. To gain insight into how RecQ4 dysfunction leads to these pathologies, several groups have used the Saccharomyces cerevisiae RecQ4 homolog Hrq1 as an experimental model. Hrq1 displays many of the same functions as RecQ4 in vivo and in vitro. However, there is some disagreement in the literature about the effects of single-stranded DNA (ssDNA) length on Hrq1 helicase activity and the ability of Hrq1 to anneal complementary ssDNA oligonucleotides into duplex DNA. Here, we present a side-by-side comparison of Hrq1 and RecQ4 helicase activity, demonstrating that in both cases, long random-sequence 3' ssDNA tails inhibit DNA unwinding in vitro in a length-dependent manner. This appears to be due to the formation of secondary structures in the random-sequence ssDNA because Hrq1 preferentially unwound poly(dT)-tailed forks independent of ssDNA length. Further, RecQ4 is capable of ssDNA strand annealing and annealing-dependent strand exchange, but Hrq1 lacks these activities. These results establish the importance of DNA sequence in Hrq1 helicase activity, and the absence of Hrq1 strand annealing activity explains the previously identified discrepancies between S. cerevisiae Hrq1 and human RecQ4.

Borrowing nuclear DNA helicases to protect mitochondrial DNA.

In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer's disease, premature aging and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases.

RECQ4 selectively recognizes Holliday junctions.

The RECQ4 protein belongs to the RecQ helicase family, which plays crucial roles in genome maintenance. Mutations in the RECQ4 gene are associated with three insidious hereditary disorders: Rothmund-Thomson, Baller-Gerold, and RAPADILINO syndromes. These syndromes are characterized by growth deficiency, radial ray defects, red rashes, and higher predisposition to malignancy, especially osteosarcomas. Within the RecQ family, RECQ4 is the least characterized, and its role in DNA replication and repair remains unknown. We have identified several DNA binding sites within RECQ4. Two are located at the N-terminus and one is located within the conserved helicase domain. N-terminal domains probably cooperate with one another and promote the strong annealing activity of RECQ4. Surprisingly, the region spanning 322-400aa shows a very high affinity for branched DNA substrates, especially Holliday junctions. This study demonstrates biochemical activities of RECQ4 that could be involved in genome maintenance and suggest its possible role in processing replication and recombination intermediates.

RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in Xenopus egg extracts.

Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase α and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.