# Lifestyle of the SCCmec element and mechanisms of self-loading helicases

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2020 · $440,407

## Abstract

Project Summary/Abstract:
 This work has two overall goals: (1) to understand the lifestyle of the mobile genetic elements (SCCs)
that carry methicillin resistance in S. aureus, and (2) to understand the mechanism of self-loading initiator
helicases, using as model systems ones that are encoded by SCC elements and their homologs from a
different family of mobile elements, the SaPIs. We will address these with a combination of biochemical and
structural tools, and in collaboration with Dr. José Penadés, microbial genetics.
 Despite the medical relevance of SCC elements, very little is known about their molecular biology
beyond the site-specific recombinases that integrate and excise them into / out of the host chromosome: no
other core “housekeeping” genes have been characterized. By examining numerous SCC elements, we
defined two patterns of conserved ORFs surrounding the recombinases. Our analysis of their sequences
suggests that they are novel replication modules.
 Both patterns include a putative helicase with a homolog among the replication initiator (“Rep”) proteins
of the staphylococcal pathogenicity islands (SaPIs). The SaPIs are an otherwise-unrelated family of mobile
genetic elements that are better characterized than the SCCs and are known to replicate after excision. The
best-studied SaPI Rep, that of SaPIBov1, is a self-loading helicase: it recognizes and opens a bubble in an
origin of replication in dsDNA, and has ATP-dependent helicase activity. We found that the SaPIBov1 Rep
homolog from SCCmec type IV is an active helicase and determined its crystal structure. Surprisingly, the
closest structural homolog to its ATPase domain is MCM, the archaeal / eukaryotic replicative helicase.
Because these Rep proteins are easy to work with, they are excellent systems for asking how self-loading
helicases morph from binding dsDNA to forming a ring around a single strand, and for understanding the
mechanism of MCM-type AAA+ helicases as well.
 Aim 1 asks are the putative replication proteins of SCC elements functional and what exactly do
they do? Preliminary results suggest that as well as the putative initiator helicases, these include novel SSBs
and a minimalist PolA family polymerase that may be a primase. We will continue use biochemical tools to
work out their in vitro activities and interactions. Our collaborator Dr. Penadés will test their proposed functions
in vivo in S. aureus. (No funds are requested for Dr. Penadés).
 Aim 2 asks How do self-loading initiator helicases work? We will use our existing crystal structure
in conjunction with electron microscopy to understand how these enzymes interact with ssDNA in helicase
mode. To understand the process of bubble opening, we will combine our existing structure with DNA
footprinting, DNA topology, other biochemistry and electron microscopy to model the complex that we propose
is two hexamers bound to ~300bp of DNA, before and after bubble formation.

## Key facts

- **NIH application ID:** 9923690
- **Project number:** 5R01GM121655-04
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** PHOEBE A RICE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $440,407
- **Award type:** 5
- **Project period:** 2017-09-01 → 2021-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9923690

## Citation

> US National Institutes of Health, RePORTER application 9923690, Lifestyle of the SCCmec element and mechanisms of self-loading helicases (5R01GM121655-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9923690. Licensed CC0.

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