PolA2 is required for embryo development in ArabidopsisThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

Botany ◽  
2009 ◽  
Vol 87 (6) ◽  
pp. 626-634 ◽  
Author(s):  
Hui Yang ◽  
Daoquan Xiang ◽  
Sathya Prakash Venglat ◽  
Yongguo Cao ◽  
Edwin Wang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Embryo Development ◽  
Dna Polymerase ◽  
Apical Cell ◽  
National Research Council ◽  
Model Systems ◽  
Proper Function ◽  
Dna Polymerase Α ◽  
Insertional Inactivation ◽  
Embryo Lethality

DNA replication machinery is highly conserved in eukaryotes. DNA polymerase is essential for the synthesis of new DNA strands and for DNA repair. Despite the significant progress in the understanding of these processes in yeast and animal model systems, there is only scant information available for their counterparts in plants. Among different multisubunit-containing DNA polymerases, DNA polymerase α (POLA complex) is composed of four subunits. In this study, we report on the characterization of PolA2, which encodes the putative B subunit of DNA polymerase α in Arabidopsis thaliana (L.) Heynh. PolA2 is a single copy gene in Arabidopsis and shows highly conserved regions with putative homologs in other plant species. Insertional inactivation of PolA2 in Arabidopsis leads to embryo lethality, with developmental arrest at or before the four-cell stage during embryogenesis. The apical cell lineage is strongly affected in the mutant embryos and the endosperm initial cell fails to divide. PolA2 is expressed broadly in the early phases of embryo development during the period of active cell divisions, while during the later stages of development expression is reduced and more localized. Ectopic overexpression of PolA2 produced dominant negative phenotypes with gametic and embryo lethality suggesting that coordinated and parallel expression with other subunits is critical for its proper function in DNA replication and plant development.

scholarly journals Effect of Dehydroaltenusin-C12 Derivative, a Selective DNA Polymerase α Inhibitor, on DNA Replication in Cultured Cells

Molecules ◽  
2008 ◽  
Vol 13 (12) ◽  
pp. 2948-2961 ◽  
Author(s):  
Isoko Kuriyama ◽  
Takeshi Mizuno ◽  
Keishi Fukudome ◽  
Kouji Kuramochi ◽  
Kazunori Tsubaki ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Cultured Cells ◽  
Dna Polymerase Α

scholarly journals A Coordinated Temporal Interplay of Nucleosome Reorganization Factor, Sister Chromatin Cohesion Factor, and DNA Polymerase α Facilitates DNA Replication

2004 ◽  
Vol 24 (21) ◽  
pp. 9568-9579 ◽  
Author(s):  
Yanjiao Zhou ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
S Phase ◽  
Terminal Region ◽  
Replication Complex ◽  
Dna Polymerase Α ◽  
Chromatid Cohesion ◽  
Phase Progression

ABSTRACT DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase α (Polα). The catalytic subunit of budding yeast Polα (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polα (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polα protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

scholarly journals The N-Terminal Noncatalytic Region of Xenopus RecQ4 Is Required for Chromatin Binding of DNA Polymerase α in the Initiation of DNA Replication

2006 ◽  
Vol 26 (13) ◽  
pp. 4843-4852 ◽  
Author(s):  
Kumiko Matsuno ◽  
Maya Kumano ◽  
Yumiko Kubota ◽  
Yoshitami Hashimoto ◽  
Haruhiko Takisawa
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Sequence Similarity ◽  
Dna Polymerases ◽  
Helicase Domain ◽  
Chromatin Binding ◽  
Replication Machinery ◽  
Dna Polymerase Α ◽  
Initiation Of Dna Replication ◽  
Replication Activity

ABSTRACT Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases α and ε onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase α. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.

Regulation of eukaryotic DNA replication at the initiation step

2003 ◽  
Vol 31 (1) ◽  
pp. 266-269 ◽  
Author(s):  
S. Pollok ◽  
J. Stoepel ◽  
C. Bauerschmidt ◽  
E. Kremmer ◽  
H.-P. Nasheuer
Keyword(s):  
Dna Replication ◽  
Cell Growth ◽  
Dna Polymerase ◽  
Molecular Control ◽  
Dna Polymerase Α ◽  
Late Protein ◽  
Two Factors ◽  
Eukaryotic Dna ◽  
A Cell ◽  
The Stability

The studies of cell growth and division have remained at the centre of biomedical research for more than 100 years. The combination of genetic, biochemical, molecular and cell biological techniques recently yielded a burst in what is known of the molecular control of cell growth processes. The initiation of DNA replication is crucial for the stability of the genetic information of a cell. Two factors, Cdc45p (cell division cycle 45p) and DNA polymerase α-primase, are necessary in this process. Depending on growth signals, Cdc45p is expressed as a late protein. New phosphorylation-specific antibodies specifically recognize the phosphorylated subunit, p68, of the four subunit DNA polymerase α-primase and show that the phosphorylated polypeptide is exclusively nuclear.

scholarly journals Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

2001 ◽  
Vol 75 (18) ◽  
pp. 8569-8578 ◽  
Author(s):  
Armin R. Kautz ◽  
Klaus Weisshart ◽  
Annerose Schneider ◽  
Frank Grosse ◽  
Heinz-Peter Nasheuer
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Complexes ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Specific Factor ◽  
Dna Polymerase Α

ABSTRACT Although p48 is the most conserved subunit of mammalian DNA polymerase α-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.

scholarly journals The B-Subunit of DNA Polymerase α-Primase Associates with the Origin Recognition Complex for Initiation of DNA Replication

2004 ◽  
Vol 24 (17) ◽  
pp. 7419-7434 ◽  
Author(s):  
Masashi Uchiyama ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Dna Polymerase ◽  
Origin Recognition Complex ◽  
Replication Origins ◽  
Dna Polymerase Α ◽  
B Subunit ◽  
Initiation Of Dna Replication ◽  
Primase Complex ◽  
Origin Recognition

ABSTRACT The B-subunit (p70/Pol12p) of the DNA polymerase α-primase (Polα-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polα-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polα-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polα-primase in the initiation of both leading and lagging strands at the replication origins.

Sign in / Sign up

Export Citation Format

Share Document