DOI: 10.1073/pnas.211342798. Functional analysis of mutant and wild-type <i>Drosophila</i> origin recognition complex

Functional analysis of mutant and wild-type Drosophila origin recognition complex

10.1073/pnas.211342798

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro , ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA + nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.

Bibliography

Chesnokov, I., Remus, D., & Botchan, M. (2001). Functional analysis of mutant and wild-type Drosophila origin recognition complex. Proceedings of the National Academy of Sciences, 98(21), 11997â12002.

Authors 3

Igor Chesnokov (first)
Dirk Remus (additional)
Michael Botchan (additional)

References 36 Referenced 94

10.1016/S0960-9822(01)00196-8
10.1146/annurev.biochem.69.1.829
10.1038/357128a0
J L Campbell, C S Newlon Chromosomal DNA Replication (Cold Spring Harbor Lab. Press, Plainview, NY, 1991). / Chromosomal DNA Replication by Campbell J L (1991)
10.1073/pnas.96.6.2656
10.1016/S0959-437X(98)80141-X
10.1101/gad.13.20.2619
10.1016/S0092-8674(01)00334-8
10.1101/gad.822101
10.1128/MCB.20.15.5581-5591.2000
10.1101/gr.9.1.27
10.1016/S0092-8674(00)81889-9
10.1101/gad.13.10.1289
S K Dhar, A Dutta J Biol Chem 201, 279 (2000). / J Biol Chem by Dhar S K (2000)
10.1073/pnas.96.22.12367
10.1074/jbc.M102493200
10.1126/science.8266075
10.1128/MCB.17.12.7159
10.1126/science.270.5242.1674
10.1073/pnas.94.8.3888
10.1016/S0960-9822(00)00844-7
10.1016/S0896-6273(00)80752-7
10.1242/dev.128.9.1697
10.1101/gad.13.20.2639
10.1101/gad.12.5.734
10.1101/gad.13.7.827
10.1016/0092-8674(95)90081-0
10.1002/j.1460-2075.1991.tb05014.x
10.1083/jcb.106.1.1
10.1016/0092-8674(86)90622-7
10.1016/S0092-8674(00)80415-8
D T S Pak Ph.D. thesis (Univ. of California, Berkeley, 1996). / Ph.D. thesis by Pak D T S (1996)
10.1016/S1097-2765(00)00062-9
10.1073/pnas.131006898
10.1093/emboj/18.9.2435
10.1038/35082600

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:34 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 8:48 a.m.)
Indexed	1 month, 4 weeks ago (July 2, 2025, 2:31 p.m.)
Issued	23 years, 10 months ago (Oct. 2, 2001)
Published	23 years, 10 months ago (Oct. 2, 2001)
Published Online	23 years, 10 months ago (Oct. 2, 2001)
Published Print	23 years, 10 months ago (Oct. 9, 2001)

Funders 0

None

BibTeX

@article{Chesnokov_2001, title={Functional analysis of mutant and wild-type Drosophila origin recognition complex}, volume={98}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.211342798}, DOI={10.1073/pnas.211342798}, number={21}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Chesnokov, Igor and Remus, Dirk and Botchan, Michael}, year={2001}, month=oct, pages={11997–12002} }

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