Replication initiation sites

In anaphase and during Cl, a prereplication complex (pre-RC) is formed at replication initiation sites which contains the Cdc6 protein and the MCM proteins, in addition to the constitutively bound origin recognition complex (ORC). Formation of pre-RC is negatively regulated by the C2/M cyclin -CDK complex. The Cdc6 protein and the MDM proteins are removed at the Cl-S transition due to phosphorylation by the S-phase cyclin - CDK complex which allows the onset of DNA replication. The activity of the cyclin-CDK complexes is con-... 

In 4>X174, however, replication begins with a single stranded closed circle, a rather atypical situation. First, primase brings about the synthesis of a short RNA primer, beginning at one or more specific initiation sites on the DNA. 

F. D. Araujo, J. D. Knox, S. Ramchandani, R. Pelletier, P. Bigey, G. Price, M. Szyf, and M. Zannis-Hadjopoulos, Identification of initiation sites for DNA replication in the human dnmtl (DNA-methyltransferase) locus. J. Biol. Chem. 274, 9335-9341 (1999). 

A main control of DNA replication occurs at the level of initiation of replication. The replication of a DNA sequence starts at specific sequence sections of the DNA, known as replication origins. In yeast, the initiation sites of DNA replication have been very well defined and characterized at the sequence level. In higher eucaryotes, in contrast, initiation has a broad initiation zone and it has not been possible to identify a defined initiation sequence. The size of the genome in eucaryotes necessitates the use of many replication origins, which can be activated in a defined time sequence and position-specific order. 

Initiation of a second round of replication leads to a replication eye at the initiation site of replication (fig. 26.3). As synthesis proceeds the size of the replication eye becomes larger at this stage the replicating chromosome is referred to as a theta structure because it has the appearance of the Greek letter d. Semiconservative replication is consistent with the density of the autoradiographic tracks made by different parts of the chromosome after one and two rounds of replication in [3H]thymidine (see fig. 26.3). 

Two aspects of E. coli chromosomal replication still to be considered are initiation and termination. From what has been said we conjecture that replication initiates at a unique site, proceeds bidirectionally, and terminates at a point where the two oppositely advancing growth forks meet. To study initiation it was first necessary to isolate that segment... 

Genetic studies have established that initiation of replication occurs at a fixed site, called the initiation site or origin of the chromosome (oriC). The nucleotide sequence in this region binds to various proteins to initiate the two forks. 

The immune system plays a central role in the pathogenesis of prion infection. In the acquired human prion diseases, it is the initial site of agent replication prior to entry into the nervous system but the immune system does not appear to be essential in the pathogenesis of the familial and sporadic human prion diseases. Followdng peripheral exposure to the prion agent, the immune system can amplify prion infection but, interestingly, the adaptive immune response does not play a role in the clearance of the prion agent. 

Like replication, transcription requires separation of the duplex DNA strands and uses a polymerase to copy the template DNA strand. For transcription, the polymerase is RNA polymerase II, which binds to sequences in the regulatory region of the gene called the promoter. Promoters occur approximately 100 bases upstream (i.e., at the 5 end) from the initiation site of transcription where the first ribonucleotide unit is paired with the template (uracil pairs with adenine). Promoters are usually rich in thymine and adenine in repeating patterns and have been referred to as a TATA box. Initiation of transcription requires many protein cofactors to bind to RNA polymerase to form the active initiation complex. Other regions of DNA known as enhancers may interact with the initiation complex to stimulate or repress transcription. Regulation of transcription is the primary mechanism cells use to control gene expression. ... 

The answer is c. (Murray, pp 412-434. Scriver, pp 3-45. Sack, pp 3—29. Wilson, pp 99-121.) Despite the great length of the chromosomes of eukaryotic DNA, the actual replication time is only minutes. This is because eukaryotic DNA is replicated bidirectionally from many points of origin. The hundreds of initiation sites for DNA replication on chromosomes share a consensus sequence called an autonomous replication sequence (ARS). Thus, while the process of DNA replication in mammals is similar to that in bacteria, with DNA polymerases of similar optimal temperatures and speed, the many replication forks allow for a rapid synthesis of chromosomal DNA. Proteins such as histones, which are bound to mammalian chromosomes, inhibit DNA replication or transcription. Dissociation of the protein-DNA complex (chromatin) and unwinding of DNA supercoils (followed by chromatin reassembly) is part of the replication process. 

The replication of the circular E. coli chromosome (Figure 18.5) begins at a precise initiation site referred to as oriC and proceeds in two directions. Helicases unwind the DNA duplex, two replisomes assemble, and replication proceeds outward in both directions. As the two sites of active DNA synthesis (referred to as replication forks) move farther away from each other, a replication eye forms. Because an E. coli chromosome contains one initiation site, it is considered a sin-... 

Describe the function and features of the nucleotide sequence of oriC and note that it is the unique site of bidirectional replication initiation in E. coll List the proteins that interact with the DNA in this region of the chromosome, and give the reactions they catalyze and functions they serve. 

Replication in E. coli is a bidirectional process and is described by the theta model of replication. In this model the new strands peel from the parent strand, appearing like a Greek letter theta (0). There is strong evidence to support a model with two replication forks that work in concert in opposite directions starting from a well-defined initiation site. By the time the forks meet up, a complete second copy of the genome has been made (Fig. 8-3). 

EXAMPLE 8.7 One way in which difference 1 in Example 8.6 is addressed is that, in contrast to the single initiation site for DNA replication in prokaryotes, there are multiple initiation sites, between 3 x 10 and 3 x 10 base pairs apart, on eukaryotic chromosomes. Therefore, even though replication fork movement is slower in eukaryotes than in prokaryotes (about 50 nucleotides s ), the presence of multiple sites of initiation allows chromosome replication to occur on a time scale of 10 h whereas it would take -500 h if there were only a single initiation site. 

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