A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU

During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the polα-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase.

Time-dependent pleiotropic phenotypes are caused by progressive DNA polymerase α deficiency in zebrafish

The mechanisms underlying variation in cell- and tissue-specific phenotypes caused by mutations are poorly understood. For example, why some cancers show cellular atypia more than others is unclear. As an illustration of phylogenetic variation in phenotype, mutations in a subunit of DNA polymerase α (Pol α), one of the primary eukaryotic replicative DNA polymerases, result in immediate cell cycle arrest in yeast and Arabidopsis; a null mutation in the same subunit results in lethality in 5 to 7 days in zebrafish, in association with striking tissue-specific, time-dependent pleiotropic cellular phenotypes. Here, we describe a zebrafish null mutant in the gene encoding the B subunit of Pol α, huli hutu (hht), in which cell death with nuclear fragmentation is found in the central nervous system where the largest number of cells arise, while cells present in smaller numbers, such as the gastrointestinal epithelial cells, instead show cellular atypia that is associated with hepatic dysgenesis and pancreatic and air bladder agenesis. Wild-type maternal mRNA in genotypically mutant eggs becomes undetectable by 24 hours post-fertilization (hpf). Furthermore, the treatment of wild-type larvae with DNA synthesis inhibitors aphidicolin or hydroxyurea from 24 hpf phenocopies hht. Taken together, the data are consistent with a model in which reduced capacity for cell proliferation caused by either genetic or chemical mechanisms of DNA synthesis beginning at about 24 hpf results in cell death in tissues with large cell number, and abnormal nuclear morphology in tissues with fewer cells, including the gastrointestinal tract.

DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication–linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.

Plant DNA polymerases alpha and delta mediate replication of geminiviruses

ABSTRACTGeminiviruses are causal agents of devastating diseases in crops. Geminiviruses have circular single-stranded (ss)DNA genomes that are replicated in the nucleus of the infected plant cell through double-stranded (ds)DNA intermediates by the plant’s DNA replication machinery; which host DNA polymerase mediates geminiviral multiplication, however, has so far remained elusive. Here, we show that subunits of the nuclear replicative DNA polymerases α and δ physically interact with the geminivirus-encoded replication enhancer protein, C3, and are required for viral replication. Our results suggest that while DNA polymerase α is essential to generate the viral dsDNA intermediate, DNA polymerase δ mediates the synthesis of new copies of the geminiviral ssDNA genome, and that the virus-encoded C3 acts selectively recruiting DNA polymerase δ over ε to favour a productive replication.

The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box

A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.