# Structural mechanism of DNA replication

> **NIH NIH R35** · VAN ANDEL RESEARCH INSTITUTE · 2024 · $720,000

## Abstract

Project Summary
DNA replication is thought to have evolved twice, independently, in Bacteria and Archaea/Eukarya, as the
principal components of the replication machinery such as the replicative helicase and the DNA polymerases
are not evolutionarily related in the two branches of life. Initiation of eukaryotic DNA replication occurs in G1
phase of the cell cycle, when the initiator Cdc6 binds and activates the Origin Recognition Complex (ORC) to
recruit Cdt1-bound Mcm2-7 hexamer, thereby assembling an inactive Mcm2-7 double-hexamer on double
stranded DNA and establishing the pre-Replication Complex (pre-RC). At G1-to-S transition, the Mcm2-7
double-hexamer is converted to two active replicative helicases, the Cdc45-Mcm2-7-GINS (CMG) complexes.
The CMG helicase and the Ctf4 trimer serve as the hubs to nucleate a replisome to synthesize DNA. Advances
in in vitro reconstitutions of origin activation, leading strand and lagging strand DNA synthesis, along with the
cryo-EM resolution revolution, have made it feasible to visualize a replisome and tackle the complex replication
mechanism. In the previous funding cycle, we have elucidated how Cdc6 binds ORC to activate it and enhance
origin specificity, how ORC-Cdc6 recruits and loads Mcm2-7 onto origin DNA in a multi-step process, and how
the CMG helicase unwinds DNA duplex at a fork. We have determined the complete structures of all three
major enzymes at the replication fork – the leading strand polymerase Pol ε, the lagging strand polymerase Pol
δ, and the polymerase-primase complex Pol α. Furthermore, we have revealed entirely unexpected loading
mechanisms of the PCNA and 9-1-1 DNA clamps by the clamp loaders RFC and Rad24-RFC, respectively.
We have shown how the yeast Yta7 ATPase hexamer disrupts the nucleosome ahead of the replication fork
and how the T4 phage helicase hexamer assembles with the primase to form a functional primosome to
synthesize a 5-nucleotide RNA primer. Building on these successes, the PI proposes to continue the
collaborative and mechanistic study on the replication origin activation mechanism and the replisome
architecture. The proposed research is significant because DNA replication is central to cellular growth, and
dysregulation of replication leads to uncontrolled proliferation and tumorigenesis.

## Key facts

- **NIH application ID:** 10849426
- **Project number:** 2R35GM131754-06
- **Recipient organization:** VAN ANDEL RESEARCH INSTITUTE
- **Principal Investigator:** Huilin Li
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $720,000
- **Award type:** 2
- **Project period:** 2019-06-07 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10849426

## Citation

> US National Institutes of Health, RePORTER application 10849426, Structural mechanism of DNA replication (2R35GM131754-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10849426. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*