Janet Harwood

Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein–Barr virus

Epstein–Barr virus (EBV) replicates in its latent phase once per cell cycle in proliferating B cells. The latent origin of DNA replication, oriP, supports replication and stable maintenance of the EBV genome. OriP comprises two essential elements: the dyad symmetry (DS) and the family of repeats (FR), both containing clusters of binding sites for the transactivator EBNA1. The DS element appears to be the functional replicator. It is not yet understood how oriP‐dependent replication is integrated into the cell cycle and how EBNA1 acts at the molecular level. Using chromatin immunoprecipitation experiments, we show that the human origin recognition complex (hsORC) binds at or near the DS element. The association of hsORC with oriP depends on the DS element. Deletion of this element not only abolishes hsORC binding but also reduces replication initiation at oriP to background level. Co‐immunoprecipitation experiments indicate that EBNA1 is associated with hsORC in vivo. These results indicate that oriP might use the same cellular initiation factors that regulate chromosomal replication, and that EBNA1 may be involved in recruiting hsORC to oriP.

Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC.

Characterization of the proteins that interact with replication origins, as well as characterization of the mechanisms by which the levels and activities of these proteins are regulated during the cell cycle, is required to understand the initiation of chromosomal DNA replication in eukaryotic cells. We have previously shown that the first detectable step in the assembly of initiation complexes in vivo involves the binding of the multisubunit origin recognition complex (ORC) and the general transcription/replication factor ABF1 protein to origins. In this paper we show that ORC is present in cells at low levels, corresponding to little more than one complete complex per replication origin, indicating that in vivo origin recognition by ORC is extremely efficient. We show that this efficient recognition requires two sequence elements, the essential A element containing the ARS consensus sequence and the functionally important B1 element, both in vitro and in vivo. Moreover, we show that origin binding by ORC in vivo does not require any other functional sequence element, indicating that it occurs independently of the binding of other factors, such as ABF1. Our results suggest a model for the roles of the individual elements of yeast replication origins.

Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line.

We analyzed the nature of spontaneous mutations at the autosomal locus coding for adenine phosphoribosyltransferase in the human colorectal carcinoma cell line SW620 to establish whether distinctive mutational pathways exist that might underlie the more complex genome rearrangements arising in tumor cells. Point mutations occur at a low rate in aprt hemizygotes derived from SW620, largely as a result of base substitutions at G.C base pairs to yield transversions and transitions. However, a novel pathway is evident in the form of multiple dispersed mutations in which two errors, separated by as much as 1,800 bp, fall in the same mutant gene. Such mutations could be the result of error-prone DNA synthesis occurring during normal replication or during long-patch excision-repair of spontaneously arising DNA lesions. This process could also contribute to the chromosomal instability evident in these tumor cells.

Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle

SUMMARY DNA replication is a pivotal event in the cell cycle and, as a consequence, is tightly controlled in eukaryotic cells. The initiation of DNA replication is dependent upon the completion of mitosis and upon the commitment to complete the cell cycle made during G1. Characterisation of the protein factors required for initiating DNA replication is essential to understand how the cell cycle is regulated. Recent results indicate that initiation complexes assemble in multiple stages during the cell cycle. First, origins are bound by the multi-subunit origin recognition complex (ORC) which is essential for DNA replication in vivo. ORC, present at little more than one complete complex per replication origin, binds to origins immediately after initiation in the previous cell cycle. ORC binding occurs by the recognition of a bipartite sequence that includes the essential ARS consensus sequence (ACS) and the functionally important B1 element adjacent to the ACS. A novel pre-replicative complex (pre-RC) assembles at origins at the end of mitosis in actively cycling cells and remains at origins until DNA replication initiates. Finally, Dbf4, which is periodically synthesised at the end of G1, interacts with replication origins. Dbf4-origin interaction requires an intact ACS strongly suggesting that interaction occurs through ORC. Dbf4 interacts with and is required for the activation of the Cdc7 protein kinase and together, Dbf4 and Cdc7 are required for the G1-S transition. Separate regions of Dbf4 are required for Cdc7- and origin-interaction suggesting that Dbf4 may act to recruit Cdc7 to replication origins where phosphorylation of some key component may cause origin firing.

Deletion mapping of highly conserved transcribed sequence downstream from APRT locus.

To investigate the nature of DNA sequence rearrangements occurring in a highly malignant human colorectal carcinoma cell line (SW620) exhibiting a high level of chromosome instability, we characterized the molecular basis of deletions eliminatingAPRT. Deletions in SW620 resembled those in a variety of cell lines. They were joined at regions of little similarity through mono-, di-, or trinucleotide repeats. Breakpoint regions were rich in di- and trinucleotide repeats that might constitute pause sites for the replication complex. Deletions ranged in size from 1.8 to ∼70 kb and were “directional” in that they eliminated sequences upstream ofAPRT but not downstream. Analysis of downstream sequences suggested that this pattern of deletion was due to the presence of another gene. Transcripts from these two genes converged but did not overlap. Given that this gene was not deleted in any hamster or human mutants, it appears essential for cell viability. This organization has important consequences for the pattern of mutation and repair of this region.

Selection of Cells Defective in Pyrimidine (TK − ) and Purine (APRT − and HPRT − Salvage

The usefulness of the purine and pyrimidine salvage pathways in the study of the mechanisms of mutation and in the selection of cell lines stably transformed by vectors expressing these genes is well documented. Unfortunately, many investigators are deterred from selecting new host strains deficient in these enzymes because of the difficulties inherent in isolating recessive mutations of autosomal genes. Furthermore, considerable suspicion was cast over somatic cell genetics by the so-called epigenetic nature of some phenotypic changes (1). However, given the clear molecular basis of the vast majority of mutant phenotypes, such apprehensions are largely unwarranted, provided that careful, clean selections are employed (e.g., see. 2).