Dataset Information

The genetic and biochemical basis of human leading strand synthesis

ABSTRACT: The maintenance of genome stability requires efficient leading strand synthesis by DNA Polymerase Epsilon (Pol). By performing CRISPR genetic screens in cells lacking the POLE4 subunit of Pol we define a genetic map of the factors required to support Pol function in the absence of its accessory subunits. A set of genes involved in iron metabolism emerge as required to sustain Iron Sulphur Cluster (ISC)-dependent Pol activity. We then dissect a synthetic lethal interaction between POLE3-POLE4 and the CHTF18-RFC2/5 complex. By combining cell biology, structural modelling and biochemistry, we define the existence of two tiers of regulation of Pol processivity: leading strand-specific loading of PCNA by CHTF18-RFC2/5 and “gripping” of newly synthesised dsDNA by POLE3-POLE4. The combined loss of these functions is incompatible with leading strand synthesis and viability. In summary, we describe the biochemical basis of human leading strand synthesis and the consequence of its dysfunction in genome stability.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Sarah Maslen

LAB HEAD: Dr. Roberto Bellelli

PROVIDER: PXD070472 | Pride | 2026-01-19

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 12

Publications

The genetic and biochemical basis of human leading strand synthesis.

Agnarelli Alessandro A Buckley-Benbow Lauryn L Ozgencil Meryem M Lad Melanie M Ampah Khamal Kwesi KK Kalinka Alex A Belan Ondrej O Maslen Sarah S Skehel Mark J MJ Walter David D Day Matthew M Bellelli Roberto R

Nature communications 20251204 1

The maintenance of genome stability requires efficient leading strand synthesis by DNA Polymerase Epsilon (Polε). By performing CRISPR genetic screens in cells lacking the POLE4 subunit of Polε we define a genetic map of the factors required to support Polε function in the absence of its accessory subunits. A set of genes involved in iron metabolism emerge as required to sustain Iron Sulphur Cluster (ISC)-dependent Polε activity. We then dissect a synthetic lethal interaction between POLE3-POLE4 ...[more]

PMID: 41339636

Similar Datasets

Project description:Faithful duplication of DNA is essential for the maintenance of genomic stability in all organisms. DNA synthesis proceeds bi-directionally with continuous synthesis of leading strand DNA and discontinuous synthesis of lagging strand DNA. Herein, we describe a method of enriching and Sequencing of Protein-Associated Nascent strand DNA (eSPAN) to detect whether a protein binds the leading- and lagging-strands of DNA replication forks. We show that Pol-epsilon, PCNA, Cdc45, Mcm6 and Mcm10 preferentially associate with leading strands, whereas Pol-alpha, Pol32, Pol-delta, Rfa1 and Rfc1 associate with lagging strands of hydroxyurea (HU)-stalled replication forks. In contrast, PCNA is enriched at lagging strands of normal replication forks in wild type cells and HU-stalled forks in cells lacking Elg1. These studies demonstrate a strategy to reveal proteins at leading and lagging strands of DNA replication forks, and suggest that the unloading of PCNA from lagging strands of HU-stalled replication forks helps maintain genome integrity. We synchronized yeast cells at G1 and released into early S phase in the presence of BrdU, a nucleotide analog that can be incorporated into newly synthesized DNA strand, and hydroxyurea (HU), a ribonucleotide reductase inhibitor. HU has no effect on initiation of DNA replication at early replication origins, but inhibit late replication firing. In addition, replication forks are stalled due to depletion of dNTPs. We then performed chromatin-immunoprecipitation of 12 proteins of interest following a standard procedure. Protein-bound DNAs were then reverse-crosslinked and double strand DNA was denatured. Nascent DNA was enriched by immunoprecipitation using anti-BrdU antibodies. The recovered ssDNA was first marked with ligation to one oligo at 3M-bM-^@M-^Y end before conversion to dsDNA for library preparation and sequencing. In this way, the directionality of ssDNA and therefore strand information of each sequenced DNA were known. The sequencing tag was mapped to both Watson (red) and Crick (blue) strands of the reference genome. In addition to ChIP-eSPAN, we also performed BrdU-IP and single strand DNA sequence (BrdU-ssSeq) and protein ChIP followed by single-strand DNA sequencing (ChIP-ssSeq) for each corresponding ChIP-eSPAN experiment. We also performed Mcm4 and Mcm6 ChIP-seq using cells synchronized at G1 phase of the cell cycle for identification of replication origins in comparison with published dataset. Some protein ChIP-ssSeq and ChIP-eSPAN experiments were repeated and the data fits well each other. Therefore, we did not repeat all protein ChIP-ssSeq and ChIP-eSPAN experiments.

			Action	DRS
	AL3286_PolE_10_1.raw	Raw
	AL3286_PolE_10_2.raw	Raw
	AL3286_PolE_20_1.raw	Raw
	AL3286_PolE_20_2.raw	Raw
	AL3286_PolE_30_1.raw	Raw