Role and Regulation of the RECQL4 Family during Genomic Integrity Maintenance

RECQL4 is a member of the evolutionarily conserved RecQ family of 3’ to 5’ DNA helicases. RECQL4 is critical for maintaining genomic stability through its functions in DNA repair, recombination, and replication. Unlike many DNA repair proteins, RECQL4 has unique functions in many of the central DNA repair pathways such as replication, telomere, double-strand break repair, base excision repair, mitochondrial maintenance, nucleotide excision repair, and crosslink repair. Consistent with these diverse roles, mutations in RECQL4 are associated with three distinct genetic diseases, which are characterized by developmental defects and/or cancer predisposition. In this review, we provide an overview of the roles and regulation of RECQL4 during maintenance of genome homeostasis.

Hells Promotes Transcription in Lymphoma By Resolving Topological Conflicts and By Preventing DNA Damage

T cell lymphomas (TCLs) are a rare and heterogeneous group of non Hodgkin Lymphoma with aggressive courses and prognoses. TCLs are characterized by genomic instability and massive transcriptional activity that is required to sustain cancer cell proliferation. Indeed, high rate of transcription increases DNA topological tension resulting in accumulation of DNA-RNA hybrid structures called R-Loops, in which the nascent RNA pairs with its DNA-template, favouring structural alterations and DNA-lesions. DNA helicases are a class of enzyme which major function is to alleviate DNA topological stresses by solving R-Loops and protecting DNA from damage. DNA helicases also facilitate RNA-PolymeraseII (RNAPII) progression along the gene body during transcription thus being the major determinant of gene expression. We recently identified the DNA-helicase HELLS - a member of the SWI/SNF2 family- as a new genetic vulnerability of TLCs. HELLS orchestrates a transcriptional program essential to the survival and proliferation of TCLs and its genetic ablation profoundly impairs mitosis and cell proliferation. To understand how HELLS manages and coordinates transcription and to define the HELLS-associated gene expression program in TCLs, we performed RNA-seq in TLBR-2 after the depletion of HELLS (HELLS KD). Gene expression profiling showed that HELLS KD affects the expression of 728 genes (413 downregulated and 315 upregulated genes). These genes were mainly involved in the regulation of cytoskeleton, cell cycle, cytokinesis, chromatin remodelling and DNA repair as indicated by a gene ontology analysis. Next, to evaluate the distribution of chromatin markers after HELLS KD, we performed Chromatin Immunoprecipitation (ChIP) sequencing against active (H3K4me3) and repressive (H3K9me3) histone markers. Depletion of HELLS results in a modest but significant change in the H3K4me3 level at promoters and distal intergenic regions. Differential analysis identified 1,571 bound sites corresponding to 1,278 genes deregulated after HELLS KD. Notably, 59/1,278 genes resulted concordantly deregulated in RNA-seq analysis. Accordingly with the role of HELLS in T lymphocytes development, gene ontology analysis on identified 59 genes reveals enrichment in T cell activation, T-helper 17 differentiation and lineage commitment processes. Instead, no significant changes in H3K9me3 level at examined regulatory regions were observed after HELLS KD, suggesting that HELLS does not modify the distribution of this marker. To deeply study the transcriptional function of HELLS, we investigated its ability to promote transcription by solving DNA topological tension. By performing S9.6 antibody staining on a panel of TCL cells, we showed that loss of HELLS leads to an accumulation of R-Loops that co-localized with the active form of RNAPII (S2-CTD), suggesting that HELLS alleviates RNAPII stall upon collision with R-loops during elongation. Co-immunoprecipitation (Co-IP) experiments showed that HELLS interacts with RNAPII and Chromatin Immunoprecipitation (ChIP) assays in TLBR-2 cells demonstrating that the loss of HELLS leads to changes in the distribution of active form of RNAPII that accumulates on transcriptional starting sites of selective target genes. By immunofluorescence staining, we detected that the decreased RNAPII activity after HELLS KD was associated with a significant decrease in the incorporation of 5-ethynyl uridine (EU) into nascent RNA confirming that transcription process is attenuated across all transcriptome. As R-loops accumulation and persistence is strictly associated with DNA damage, we assessed by immunofluorescences, the level of yH2AX (marker of DNA-damage) upon depletion of HELLS. We observed a significant increase in the intensity of nuclear yH2AX signal and a formation of yH2AX foci in TCL HELLS KD cell lines. Noticeably, accumulating yH2AX foci were observed in correspondence of R-loops and in co-localization with the active form RNAPII (S2-CTD). Collectively, our results indicate that HELLS plays an essential function in supporting TCLs progression promoting gene expression by resolving DNA topological conflicts, easing RNAPII progression and protecting DNA from damaging events simultaneously. These key functions qualify HELLS as a new dependency of TCLs and therefore as potential vulnerability of these lymphomas. Disclosures No relevant conflicts of interest to declare.

Natural variation identifies SNI1, the SMC5/6 component, as a modifier of meiotic crossover in Arabidopsis

The frequency and distribution of meiotic crossovers are tightly controlled; however, variation in this process can be observed both within and between species. Using crosses of two natural Arabidopsis thaliana accessions, Col and Ler, we mapped a crossover modifier locus to semidominant polymorphisms in SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), which encodes a component of the SMC5/6 complex. The sni1 mutant exhibits a modified pattern of recombination across the genome with crossovers elevated in chromosome distal regions but reduced in pericentromeres. Mutations in SNI1 result in reduced crossover interference and can partially restore the fertility of a Class I crossover pathway mutant, which suggests that the protein affects noninterfering crossover repair. Therefore, we tested genetic interactions between SNI1 and both RECQ4 and FANCM DNA helicases, which showed that additional Class II crossovers observed in the sni1 mutant are FANCM independent. Furthermore, genetic analysis of other SMC5/6 mutants confirms the observations of crossover redistribution made for SNI1. The study reveals the importance of the SMC5/6 complex in ensuring the proper progress of meiotic recombination in plants.

Examination of the roles of a conserved motif in the PriA helicase in structure-specific DNA unwinding and processivity

DNA replication complexes (replisomes) frequently encounter barriers that can eject them prematurely from the genome. To avoid the lethality of incomplete DNA replication that arises from these events, bacteria have evolved “DNA replication restart” mechanisms to reload replisomes onto abandoned replication forks. The Escherichia coli PriA DNA helicase orchestrates this process by recognizing and remodeling replication forks and recruiting additional proteins that help to drive replisome reloading. We have identified a conserved sequence motif within a linker region of PriA that docks into a groove on the exterior of the PriA helicase domain. Alterations to the motif reduce the apparent processivity and attenuate structure-specific helicase activity in PriA, implicating the motif as a potential autoregulatory element in replication fork processing. The study also suggests that multiple PriA molecules may function in tandem to enhance DNA unwinding processivity, highlighting an unexpected similarity between PriA and other DNA helicases.

An emerging picture of FANCJ’s role in G4 resolution to facilitate DNA replication

A well-accepted hallmark of cancer is genomic instability, which drives tumorigenesis. Therefore, understanding the molecular and cellular defects that destabilize chromosomal integrity is paramount to cancer diagnosis, treatment and cure. DNA repair and the replication stress response are overarching paradigms for maintenance of genomic stability, but the devil is in the details. ATP-dependent helicases serve to unwind DNA so it is replicated, transcribed, recombined and repaired efficiently through coordination with other nucleic acid binding and metabolizing proteins. Alternatively folded DNA structures deviating from the conventional anti-parallel double helix pose serious challenges to normal genomic transactions. Accumulating evidence suggests that G-quadruplex (G4) DNA is problematic for replication. Although there are multiple human DNA helicases that can resolve G4 in vitro, it is debated which helicases are truly important to resolve such structures in vivo. Recent advances have begun to elucidate the principal helicase actors, particularly in cellular DNA replication. FANCJ, a DNA helicase implicated in cancer and the chromosomal instability disorder Fanconi Anemia, takes center stage in G4 resolution to allow smooth DNA replication. We will discuss FANCJ’s role with its protein partner RPA to remove G4 obstacles during DNA synthesis, highlighting very recent advances and implications for cancer therapy.

Phylogenetic Diversity of Lhr Proteins and Biochemical Activities of the Thermococcales aLhr2 DNA/RNA Helicase

Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.

Structural characterisation of the Chaetomium thermophilum Chl1 helicase

Chl1 is a member of the XPD family of 5’-3’ DNA helicases, which perform a variety of roles in genome maintenance and transmission. They possess a variety of unique structural features, including the presence of a highly variable, partially-ordered insertion in the helicase domain 1. Chl1 has been shown to be required for chromosome segregation in yeast due to its role in the formation of persistent chromosome cohesion during S-phase. Here we present structural and biochemical data to show that Chl1 has the same overall domain organisation as other members of the XPD family, but with some conformational alterations. We also present data suggesting the insert domain in Chl1 regulates its DNA binding.

Special Issue: DNA Helicases: Mechanisms, Biological Pathways, and Disease Relevance

DNA helicases have emerged as a prominent class of nucleic acid-metabolizing enzymes that play important roles in genome maintenance and cellular homeostasis [...]