The WYL domain of the PIF1 helicase from the thermophilic bacteriumThermotoga elfiiis an accessory single-stranded DNA binding module

Abstract

PIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N-and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its function in vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii (TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostable in vitro, and displayed activities similar to its eukaryotic homologs. We also found that the WYL domain was necessary for high-affinity Supporting Information.

Structure and functional implications of WYL-domain-containing transcription factor PafBC involved in the mycobacterial DNA damage response

“…Such a potential factor may thus well be an RNA molecule or, in a broader context, a nucleic-acid or nucleic acid derivative, relaying the signal of DNA damage to PafBC by recognition at the WYL and/or WCX domains. In fact, the WYL domain of PIF1 helicase from Thermotoga elfii was shown to bind singlestranded DNA, thereby stimulating helicase activity (Andis et al, 2018). Binding of single-stranded DNA may be conceivable for the WYL domain proteins of class J of our computational analysis, which are also associated with a helicase domain.…”

“…However, understanding and experimentation concerning the regulatory mechanism of PafBC was largely hampered by a lack of knowledge about its molecular structure. This limitation has manifested also in other studies concerning WYL domain-containing proteins (Andis et al, 2018;Hein et al, 2013;Modell et al, 2014;Yan et al, 2018). Our computational analysis showed that roughly 90% of all WYL domain-containing proteins possess an N-terminal HTH domain suggesting that these are transcriptional regulators (Figure 6a).…”

“…It has been suggested that the WYL domain might play the role of a ligand-binding domain in the context of this class of transcription factors. A handful of other WYL domain-containing proteins were studied to date: (1) DriD, an SOS responseindependent transcriptional activator of a cell division inhibitor protein in Caulobacter crescentus (Modell et al, 2014) (2) Sll7009, Sll7062, Sll7078, transcriptional repressors of CRISPR/Cas system mature crRNA in Synechocystis 6803 (Hein et al, 2013), (3) PIF1 helicase from Thermotoga elfii (Andis et al, 2018) and (4) WYL domain-containing proteins stimulating RNA cleavage by Cas13d in Eubacterium siraeum and Ruminococcus sp. (Yan et al, 2018).…”

The biochemical activities of the Saccharomyces cerevisiae Pif1 helicase are regulated by its N-terminal domain

“…We previously demonstrated that the non-helicase CTD (a predicted WYL domain [32]) of the bacterial TePif1 helicase is an accessory ssDNA binding domain [12]. This WYL domain impacts both ATP hydrolysis and DNA unwinding, coupling the two to regulate the biochemistry of the helicase.…”

“…Among the two Saccharomyces cerevisiae PIF1 family helicases, Rrm3 and Pif1, it is known that the NTD of Rrm3 is essential for its functions in vivo because truncation of the Rrm3 NTD phenocopies the null allele [11]. However, the Rrm3 NTD can be fused to the S. cerevisiae Pif1 helicase domain or the helicase domain of bacterial Pif1 proteins, and these chimeras can rescue the synthetic lethality of cells lacking Rrm3 and, for instance, the gene encoding the Srs2 helicase [12]. Thus, in this experimental set up, the PIF1 helicase domains are generic and interchangeable motor modules, while the Rrm3 NTD is vital for genome integrity.…”