Replicon

Replicon 1677 polymerase polypeptide itself, as with bacterial DNA polymerase I (Pol I), and sometimes by a separate protein, as in bacterial Pol III where the polymerase function is carried out by the a subunit and the proofreading function by the e subunit. Essentially, proofreading is the action of a 30 to 50 exonuclease which has a greater probability of excising a newly polymerized base that is mismatched than one that is correctly matched. In vitro work has shown that at least 92% of misinserted nucleotides are removed by the e subunit of Pol III, except where the next template base correctly matches the inserted mismatched base. Overall, the presence of the e subunit in bacterial Pol III typically reduces misincorporation frequencies to between 10 7 and 10 6. The efficiency of proofreading is determined in part by the polymerase that has undertaken the synthesis. Polymerase error rate is a function not only of the probability of inserting a mismatched base, but of the ability of the polymerase to use the mismatched base as a primer for further synthesis. A polymerase that is reluctant to continue synthesis on a mismatched primer terminus will allow much more time for proofreading to act than one that continues synthesis and so hides the mismatched base from the proofreading exonuclease. This property of a polymerase may be quite independent of its intrinsic misincorporation rate. Quantitatively more important than proofreading are mismatch correction processes which occur some way behind the replication fork. In these processes, enzymes remove either the incorrect base or a section of the newly synthesized strand that contains the mismatch. In bacteria the most important general mechanism is one which removes a long patch (around 103 nucleotides) of newly synthesized DNA and allows a second attempt at polymerization. Generalized mismatch correction operates not only on mismatched bases but also on small frameshifts which cause one strand to loop out. The proteins involved in this pathway are conserved from bacteria to humans and the mechanism is an important defense against cancer. To be effective, generalized mismatch correction must not only recognize the presence of a mismatch but also distinguish which strand is parental and which is newly synthesized. In E. coli this is achieved by means of a methylation tag. Soon, but not immediately, after polymerization a methyl group is attached to adenine residues at specific sequences in the DNA. Until this is done the newly synthesized strand can be recognized by the absence of the methyl groups and there is thus a window of time in which mismatch correction can take place. Other bacteria and higher organisms have other ways of distinguishing newly synthesized from parental strands of DNA, most of which remain cloaked in mystery.

The combined operation of mismatch correction processes following replication and proofreading enables the overall error frequency to be reduced to below 10 9 or less per base pair replicated. While most replicative DNA polymerases have error rates below 10 4, some specialized polymerases exist with much higher error rates. DNA polymerases IV and V in E. coli, for example, have error rates of the order of 10 4±10 5 and have specialized roles for the generation of genetic variability and for synthesizing past damage in the template strand. Other error-prone polymerases are suspected of being responsible for the somatic hypermutation that occurs in mammalian immunoglobulin genes. See also: DNA Repair; DNA Replication; Genome Size

Replication Eye Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1998

The replication eye is a region within a longer, unreplicated region, in which DNA has undergone replication. See also: Replication

Replication Fork Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1999

The replication fork is the point at which DNA strands are separated in preparation for replication. Replication forks thus move along the DNA as replication proceeds. See also: Okazaki Fragment; Origin (ori); Replication

Replicon J H Miller Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1100

A unit of replication consisting of an origin replication, a terminator on one or both sides, and the

1678

Replisome

segment of adjacent DNA under the control of the origin and terminator(s). See also: Replication

Replisome Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.2102

A replisome is a complex of proteins involved in the replication of DNA which moves along as the new complementary strand is synthesized. Main components include DNA polymerase III and a primosome. It has been suggested that an RNA replisome may be an evolutionary ancestor of the ribosome. See also: DNA Polymerases; Primosome; Replication

Reporter Gene Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.2001

A reporter gene is one that encodes an easily assayed product (e.g., chloramphenicol transacetylase) that is coupled to a promoter of interest and transfected into cells. Expression of the gene (under different conditions, or in the presence of other factors) can be used to assay promoter function. See also: Promoters

Repression Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.2002

Repression is the ability of bacteria to prevent synthesis of certain enzymes when their products are present. It is caused by inhibition of transcription or translocation by virtue of the binding of repressor protein to a specific site on DNA or mRNA. See also: Repressor

Repressor I Schildkraut Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1101

A repressor is a protein that binds to a short specific DNA sequence and controls the expression of a gene or operon. A repressor is a negatively acting regulatory protein. It binds to the operator region of a promoter and thereby negatively influences the ability of RNA polymerase to transcribe the gene or operon. The binding of a repressor to a specific DNA sequence ensures that it will not control other genes or operons and is specific for its own operator sequence. A repressor can also bind to a small molecule, which is called an effector. There are two types of effectors. One type is called an inducer. When an inducer is bound to its repressor, the repressor losses its ability to bind to its operator sequence. In the absence of the inducer, the repressor binds to its operator. The other type of effector is called a corepressor. When a small molecule corepressor is bound to its repressor, the repressor gains the ability to bind to its operator; in the absence of the corepressor, the repressor does not bind to the operator. When repressors are not bound to their cognate operator the gene or operon can be transcribed by RNA polymerase. The lac repressor of E. coli is a well-studied example of a repressor whose effector is an inducer. The lac repressor controls the expression of the lactose operon, which is responsible for the metabolism of lactose. The lactose operon is composed of three genes and all three are transcribed into a single polycistronic messenger RNA. In the absence of lactose in the medium a bacterium has no need to produce the proteins necessary for the metabolism of lactose. The lac repressor ensures that the cell will not waste resources by transcribing the lac operon. The lac repressor has two binding sites. One is specific for the operator sequence on DNA and the other is specific for the inducer, in this case, lactose. When lactose is added to the medium, lactose is transported into the cell and binds to the lac repressor. After the lac repressor binds the lactose it undergoes a slight alteration in its structure and no longer has an affinity for the lac operator. The genes necessary for the utilization of lactose are transcribed and translated. Lactose can then be utilized as a carbon source. Once the lactose is depleted, lactose no longer binds to the repressor and the repressor's structure returns to the uninduced state and binds to the lac operator blocking its transcription.