Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

Abstract

The eukaryotic genome is primarily replicated by two DNA polymerases, Pol e and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol e and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol e on the lagging strand, and CMG protects Pol e against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ-PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol e binds CMG with a K d value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ-PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ-CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.D uplication of genetic material is performed by a dynamic interplay of numerous different proteins, collectively referred to as the replisome, that orchestrate their actions to accomplish efficient, high-fidelity replication of both the leading and lagging strands (1-3). Eukaryotes possess several replisome factors that have no homologs in bacteria, and also require two different DNA polymerases for bulk leading and lagging strand synthesis, and Escherichia coli and its phages use identical copies of a DNA polymerase for both strands of the duplex (4). Thus far, the evolutionary purpose behind the additional complexity in eukaryotes is unclear.At the heart of the eukaryotic replisome is an 11-subunit helicase complex referred to as CMG (Cdc45-Mcm2-7-GINS) (5-7). Eukaryotes use three different multisubunit B-family DNA polymerases (Pol) for replication. Pol e and Pol δ copy the bulk of the genome, and Pol α-primase generates hybrid RNA-DNA primers of about 25 nucleotides and, unlike Pol e and Pol δ, it lacks 3′-5′ exonuclease (proofreading) activity. The replication factor C (RFC) clamp loader is used to load PCNA (proliferating cell nuclear antigen) clamps onto primed sites that endow the replicative polymerases with high processivity (8). The replication protein A (RPA) heterotrimer binds and protects the single-strand (ss) DNA of the lagging strand against nucleases, and helps melt secondary structure in ssDNA. Reconstitution of the core eukaryotic replisome using all thre...

Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

“…In contrast, ssDNA-binding proteins with specificity for P/T junctions should be carefully considered. An RFC·PCNA complex binds tightly to a P/T junction ( K D ~ 5 – 10 nM) 30, 31 . Hence, a ssDNA-binding protein with a comparable affinity ( K D < 50 nM) may effectively compete with an RFC·PCNA complex at modest concentrations (< 1 μM) (Figure 6B).…”

Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle

“…As discussed above in section 2 , the affinity of pol ε for PCNA encircling a P/T junction is dramatically weak compared to pol δ (see also section 4.3 below) and, hence, the contribution of the sliding clamp to continuous DNA synthesis by pol ε is relatively minimal 1, 31, 42, 43, 49–51 . In fact, cellular pol ε was initially isolated from pol δ-containing fractions and referred to as “PCNA-independent pol δ” or “pol δ 2 ” based on the marginal stimulation of its DNA synthesis activity by PCNA 50 .…”

“…Thus, pol ε and pol α are located on opposite sides of the CMG helicase. Pol δ is not stabilized on the leading strand by CMG and can be ejected by pol ε, even in the presence of PCNA 31, 48, 49 . Conversely, the affinity of pol ε for PCNA alone on the lagging strand is relatively weak such that pol ε can be ejected from PCNA by pol δ or RFC, even when the pol ε● PCNA complex is replicating DNA 1, 31, 42, 43, 49–51 .…”

“…Indeed, free, i.e. non-replicating, pol ε binds to the CMG helicase with very high affinity (low nM) in solution such that CMG helicase and pol ε can be purified from budding yeast cells as a stable protein complex 42, 49 . Furthermore, a study on X. laevis egg extracts that analyzed chromatin binding of replication fork proteins suggested that when both template DNA strands are continuous, the CMG helicase remains intact upon uncoupling from leading strand DNA synthesis and maintains contact with pol ε as the replication fork progresses 87 .…”

“…Such an extended and stiffened conformation antagonizes loop formation and is expected to strain a P/T junction●pol ε●CMG helicase complex. Furthermore, RPA is ~240-fold more abundant than pol ε in human cells and binds to ssDNA with extremely high affinity (< pM), more than 3 orders of magnitude tighter than the affinity of non-replicating pol ε for a native P/T junction 49, 101, 102 . Thus, RPA effectively outcompetes non-replicating pol ε for ssDNA.…”