Kulczyk, A. W. | Search bioRxiv

bioRxiv2026

Helicase-driven unwinding defines the architecture of the leading-strand replisome

DNA replication is a fundamental biological process that requires the coordinated activities of DNA helicase, DNA polymerase, and accessory proteins within the replisome. Although helicase-driven unwinding (HDU) has long been the accepted model for replisome progression, recent structural studies have proposed an alternative polymerase-driven unwinding (PDU) model, in which DNA polymerase physically separates duplex DNA. Here, we perform comprehensive structural and biochemical analyses to define the molecular architecture and functional interactions of the bacteriophage T7 leading-strand replisome to distinguish between these two mutually exclusive models. We show that the T7 DNA primase-helicase is positioned directly at the replication fork junction to drive DNA unwinding, whereas T7 DNA polymerase, in complex with its processivity factor E. coli thioredoxin, trails 11 to 15 nucleotides behind. Mutational analysis of T7 DNA polymerase reveals that a {beta}-hairpin previously implicated as a strand-separation pin in PDU functions instead to stabilize DNA polymerase binding to the DNA template. Furthermore, we identify an essential interaction between an acidic amino acid patch in the exonuclease domain of T7 DNA polymerase and the primase domain of T7 DNA primase-helicase, which promotes highly processive leading-strand synthesis and is compatible only with the HDU model. Together, these findings provide evidence supporting helicase-driven unwinding and clarify the molecular organization of the T7 leading-strand replisome. Because the phage T7 replisome serves as a model for DNA replication, these results have broad mechanistic implications for replisomes across taxa. Significance StatementProcessive DNA replication requires coordination between enzymes that unwind and copy DNA. Two mutually exclusive models have been proposed for replisome progression. In helicase-driven unwinding (HDU), DNA helicase separates the parental duplex at the replication fork to generate single-stranded templates for DNA synthesis. In contrast, an alternative model proposes polymerase-driven unwinding (PDU). Using structural and biochemical analyses, we investigated the architecture and functional interactions of the leading-strand replisome from bacteriophage T7. Our results show that DNA helicase performs DNA unwinding, while DNA polymerase follows to synthesize the nascent strand. We identify specific intermolecular interactions essential for leading-strand replication that are compatible only with the HDU model. These findings support helicase-driven unwinding and provide insights with broad mechanistic implications across taxa.

biochemistry↗

bioRxiv2023

YlaN is an iron(II) binding protein that functions to relieve Fur-mediated repression of gene expression in Staphylococcus aureus

Iron (Fe) is a trace nutrient required by nearly all organisms. As a result of the demand for Fe and the toxicity of non-chelated cytosolic ionic Fe, regulatory systems have evolved to tightly balance Fe acquisition and usage while limiting overload. In most bacteria, including the mammalian pathogen Staphylococcus aureus, the ferric uptake regulator (Fur) is the primary transcriptional regulator that controls the transcription of genes that code for Fe uptake and utilization proteins. YlaN was demonstrated to be essential in Bacillus subtilis unless excess Fe is added to the growth medium, suggesting a role in Fe homeostasis. Here, we demonstrate that YlaN is expendable in S. aureus; however, YlaN became essential upon Fe deprivation. A null fur allele bypassed the essentiality of YlaN. The transcriptional response of Fur derepression resulted in a reprogramming of metabolism to prioritize fermentative growth over respiratory growth. The absence of YlaN diminished the derepression of Fur-dependent transcription during Fe limitation. Bioinformatic analyses suggest that ylaN was recruited to Gram positive bacteria and once acquired was maintained in the genome as it co-evolved with Fur. Consistent with a role for YlaN in influencing Fur-dependent regulation, YlaN and Fur interacted in vivo. YlaN bound Fe(II) in vitro using oxygen or nitrogen ligands with an association constant that is consistent with a physiological role in Fe sensing and/or buffering. These findings have led to a model wherein YlaN is an Fe(II) binding protein that influences Fur-dependent regulation through direct interaction. ImportanceIron (Fe) is an essential nutrient for nearly all organisms. If Fe homeostasis is not maintained, Fe can accumulate in the cytosol where it is toxic. Questions remain about how cells efficiently balance Fe uptake and usage to prevent imbalance. Iron uptake and proper metalation of proteins are essential processes in the mammalian bacterial pathogen Staphylococcus aureus. Understanding the gene products involved in Fe ion regulation, uptake, and usage, as well as the physiological adaptations that S. aureus uses to survive in Fe-depleted conditions, will provide insight into the role that Fe has in pathogenesis. These data will also provide insight into the selective pressures imparted by the mammalian host.

microbiology↗