Telomere C-Strand Fill-In Machinery: New Insights into the Human CST-DNA Polymerase Alpha-Primase Structures and Functions.

Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.

A conserved polar residue plays a critical role in mismatch detection in A-family DNA polymerases.

In A-family DNA polymerases (dPols), a functional 3'-5' exonuclease activity is known to proofread newly synthesized DNA. The identification of a mismatch in substrate DNA leads to transfer of the primer strand from the polymerase active site to the exonuclease active site. To shed more light regarding the mechanism responsible for the detection of mismatches, we have utilized DNA polymerase 1 from Aquifex pyrophilus (ApPol1). The enzyme synthesized DNA with high fidelity and exhibited maximal exonuclease activity with DNA substrates bearing mismatches at the -2 and - 3 positions. The crystal structure of apo-ApPol1 was utilized to generate a computational model of the functional ternary complex of this enzyme. The analysis of the model showed that N332 forms interactions with minor groove atoms of the base pairs at the -2 and - 3 positions. The majority of known A-family dPols show the presence of Asn at a position equivalent to N332. The N332L mutation led to a decrease in the exonuclease activity for representative purine-pyrimidine, and pyrimidine-pyrimidine mismatches at -2 and - 3 positions, respectively. Overall, our findings suggest that conserved polar residues located towards the minor groove may facilitate the detection of position-specific mismatches to enhance the fidelity of DNA synthesis.

A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.

The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.

Primase and polymerase α tango to make an RNA-DNA hybrid primer.

Several recent cryo-electron microscopy (cryo-EM) studies about the eukaryotic primosome, including the human primosome described by Yin et al. in this issue, have uncovered the structural intricacies between the RNA primase and the DNA polymerase. These studies show that these two partners tango on DNA to synthesize a hybrid primer composed of ~ 10 nucleotide (nt) RNA and ~ 10-nt DNA. They reveal key intermediate steps involved in this process; from the self-inhibited apo state to the initiation of RNA primer synthesis, RNA primer handover to the polymerase, primer elongation by polymerase, and finally, primer termination and release. Remarkably, the polymerase domain orchestrates all major steps during primer synthesis.