sign in sign up
<<
Home , DNA polymerase II

Lecture 1

DNA REPLICATION Objectives: 1. To understand the replication process and where it occurs in the cell cycle. 2. To describe the Replication Fork, and the involved in the unwinding process and replication. 3. To understand how DNA initiate the synthesis of new strands. 4. To define and and their clinical significance.

Gene expression also called expression or often simply expression: is the process by which a gene's DNA sequence is converted into the structures and functions of a cell.

The amount of protein that a cell expresses depends on: 1. the tissue, 2. the developmental stage of the organism 3. and the metabolic or physiologic state of the cell.

DNA replication or DNA synthesis is the process of copying a double-stranded DNA strand, prior to cell division. The two resulting double strands are identical (if the replication went well), and each of them consists of one original and one newly synthesized strand. This is called semi conservative replication.

1 Prof. Dr. H.D.El-Yassin 2013 Lecture 1 The process of replication consists of three steps, initiation, replication and termination.

1. Prokaryotic replication Basic Requirement for DNA Synthesis 1. Substrates: the four deoxy nucleosides triphosphates are needed as substrates for DNA synthesis. Cleavage of the high-energy phosphate bond between the α and β phosphates provides the energy for the addition of the nucleotide. 2. Template: DNA replication cannot occur without a template. A template is required to direct the addition of the appropriate complementary deoxynucleotide to the newly synthesized DNA strand. 3. Primer: DNA synthesis cannot start without a primer, which prepares the template strand for the addition of nucleotides. 4. : the DNA synthesis that occurs during the process of replication is catalyzed by enzymes called DNA-dependent DNA . Commonly called DNA polymerases.

DNA polymerase

A DNA polymerase is an enzyme that assists in DNA replication. Such enzymes catalyze the polymerization of deoxyribonucleotides alongside a DNA strand, which they "read" and use as a template. The newly polymerized molecule is complimentary to the template strand and identical to the template's partner strand.

All DNA polymerases synthesize DNA in the 5' to 3' direction. But no known DNA polymerase is able to begin a new chain. They can only add a nucleotide onto a preexisting 3'- OH group. For this reason DNA polymerase needs a primer at which it can add the first nucleotide.

DNA polymerase I: is an enzyme that aids in DNA replication. It was discovered in the mid 1950's, and was the first such enzyme discovered (hence the name). It is often referred to as Pol I, for short. DNA polymerase I removes the RNA primer from the lagging strand and fills in the necessary nucleotides. then joins the various fragments together into a continuous strand of DNA.

DNA polymerase II: is a minor DNA polymerase in E. coli, may be involved on some DNA repair processes. It is often referred to as Pol II, for short.

DNA polymerase III holoenzyme: Pol III is a holoenzyme that aids in DNA replication. As a replicative enzymatic mechanism of DNA, the Polymerase replicates with high fidelity.

Origin of Replication

The (also called replication origin or oriC) is a unique DNA sequence at which DNA replication is initiated and proceeds bidirectionally or unidirectionally.

1. OriC: The origin of replication oriC is a 250 bp sequence rich in adenine-thymine base pairs, which are more easily separated than cytosine-guanine base pairs. 2. DnaA: dnaA is an initiation factor which hydrolyzes ATP and promotes the unwinding or melting of DNA at oriC, during DNA replication. The oriC/dnaA complex formation does not require ATP until it is open.

After initiation, dnaA binds dnaB and dnaC.

3. Replication fork: The replication fork is a structure which forms when DNA is ready to replicate itself. It is created by , which breaks the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. DNA polymerase then goes to work on creating new partners for the two strands by adding nucleotides.

2 Prof. Dr. H.D.El-Yassin 2013 Lecture 1 Basic Molecular Events at Replication Forks: 1. Leading strand synthesis: is the continuous synthesis of one of the daughter strands in a 5' to 3' direction. Pol III catalyzes leading strand synthesis.

2. Lagging strand synthesis: a. Okazaki fragments: One of the newly synthesized daughter strands is made discontinuously. The resulting short fragments are called Okazaki fragments. These fragments are latter joined by DNA ligase to make a continuous piece of DNA. This is called lagging strand synthesis. Discontinuous synthesis of lagging strands occurs because DNA synthesis always occurs in a 5' to 3' direction. Pol III catalyzes lagging strand synthesis b. Direction of new synthesis: As the replication fork moves forward, leading strand synthesis follows. A gap forms on opposite strand because it is in the wrong orientation to direct continuous synthesis of a new strand. After a lag period, the gap that forms is filled in by 5' to 3' synthesis. This means that new DNA synthesis on the lagging strands is actually moving away from the replication fork. c. Priming of Okazaki fragment synthesis. i. Enzyme: an enzyme called is the catalytic portion of a primosome that makes the RNA primer needed to initiate synthesis of Okazaki fragment. It also makes the primer that initiates leading strand synthesis at the origin. ii. Primers provide a 3'-hydroxyl group that is needed to initiate DNA synthesis. The primers made by primase are small pieces of RNA (4-12 nucleotides) complementary to the template strand. d. The role of pol I in replication: On completion of lagging strand synthesis by pol III, the RNA primer is then removed by pol I and replaced with DNA. Synthesis of each new Okazaki fragments takes place until it reach's the RNA primer of the preceding Okazaki fragment and the RNA primer. DNA pol I uses its nick-translation properties to hydrolyze the RNA (5' to 3' activity) and replace it with DNA. e. Joining of Okazaki fragments: After pol I has removed the RNA primer and replaced it with DNA, an enzyme called DNA ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent 3'OH and 5'phosphate. This can fill in the unattached gap left when the RNA primer is removed and filled in. The DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.: 3 Prof. Dr. H.D.El-Yassin 2013 Lecture 1

Other Factors Needed for Propagation of Replication Forks 1. Topoisomerase is responsible for initiation of the unwinding of the DNA. 2. : are enzymes that catalyze the unwinding of the DNA helix. A derives energy from cleavage of high energy phosphate bonds of nucleoside triphosphates, usually ATP, to unwind the DNA helix. Hilcase activity provides single strand templates for replication: 3. Gyrase. : Positive supercoils would build up in advance of a moving replication fork without the action of gyrase, which is a topomerase. 4. single-strand binding protein (SSBP): a. Function: SSBP enhances the activity of helicase and binds to a single-strand template DNA until it can serve as a template. It may also serve to protect single strand DNA from degradation by nucleases, and it may block formation of intrastrand duplexes of hairpins that can slow replication. b. Release: SSBP is displaced from single strand DNA when the DNA undergoes replication. 5. Primosome a. Definition: the primosome is a complex of that comprises primase, a hexamer of the helicase dnaB protein, dnaC protein and several other proteins. b. Function: the primosome complex primes DNA synthesis at the origin. Driven by ATP hydrolysis, the primosome moves with the replication fork, making RNA primes for Okazaki fragment synthesis. The : It is believed that all the replication enzymes and factors are part of a large macromolecular complex called replisome. It has been suggested that the replisome may be attached to the membrane and that instead of the replisome moving along the DNA during replication, DNA passed through the stationary replisome. Replosome model of replication

Termination of Replication: Replication sequences (e.g. ter) direct termination for replication. A specific protein (the termination utilization substance (TUS) protein) binds to these sequences and prevents the helicase dnaB protein from further unwinding DNA. This facilitates the termination of replication.

2. Eukaryotic Replications

Eukaryotes are organisms with complex cells, in which the genetic material is organized into membrane-bound nuclei They may utilize slightly different mechanisms of replication. However most of these mechanisms are very similar to those in prokaryotic replication.

Replicons are basic units of replication. 1. Function: A encompasses the entire DNA replicated from the growing replication forks that share a single origin. 2. Size: Replicons may vary in size from 50-120 μm. There are estimated to be 10,000-100,000 replicon per cell in mammals. The large number of replicons is needed to replicate the large mammalian genomes in a reasonable period of time. It takes approximately 8 hours to replicate the human genome. 3. Replication rate: a. . An E. coli replication fork progresses at approximately 1000 base pairs per second. b. Eukaryotes. The eukaryotic replication rate is about 10 times slower than the prokaryotic replication rate. Each replicon complete synthesis in approximately an hour. Therefore during the total period of eukaryotic replication not every replicon is active. The slow rate of eukaryotic replication is likely due to interferences of nucleusomes and chromosomal proteins. 4 Prof. Dr. H.D.El-Yassin 2013 Lecture 1

Multiple Eukaryotic DNA Polymerases

1. DNA polymerase alpha : This enzyme is composed of 4 subunits, one of which (167 kDa) carries the polymerase activity. It is responsible for synthesis of the primer on the lagging strand because it is responsible for the initiation of Okazaki fragments. The primer consists of both RNA and a short stretch (20 nt) of DNA. 2. DNA polymerase delta : This enzyme contains at least 4 and maybe as many as a dozen subunits. It has a proofreading activity. When associated with proliferating cell nuclear antigen (PCNA), it has a very high . 3. DNA polymerase beta & DNA polymerase epsilon: Both enzymes are involved in DNA repair. 4. DNA polymerase gamma: This enzyme is located in the mitochondrion where it is responsible for replication of mtDNA.

5 Prof. Dr. H.D.El-Yassin 2013 Lecture 1 and Telomerase

A telomere is a region of highly repetitive DNA at the end of a , which functions as an aglet. If it were not for telomeres, this would quickly result in the loss of useful genetic information.

In prokaryotes, are circular and thus do not have ends to suffer premature replication termination at. Only eukaryotes possess or require telomeres.

Telomeres are extended by , Telomerases are very interesting DNA polymerases in that they carry an RNA template for the telomere sequence within them.

 Structure of telomeres: In humans, the telomere sequence is a repeating string of TTAGGG, between 3 and 20 kilobases in length. There are additional 100-300 kilobases of telomere- associated repeats between the telomere and the rest of the chromosome. Telomere sequences vary from species to species, but are generally GC-rich.  The mechanism of telomere replication: Telomerase provide an RNA template complementary to the telomeric repeat, and the free 3' end of the telomere is the primer for new DNA synthesis. After elongation of the telomere by telomerase, normal lagging strand synthesis presumably makes a complementary copy of all but the 3' most terminal sequences.

In most multicellular eukaryotes, telomerase is only active in germ cells. There are theories that the steady shortening of telomeres with each replication in somatic (body) cells may have a role in senescence and in the prevention of cancer.

Clinical relevance of telomeres: If telomeres become too short, they will uncap. The cell will detect this as DNA damage and will enter cellular senescence (growth arrest). Uncapped telomeres also result in chromosomal fusions. Since this damage cannot be repaired in normal somatic cells, the cell may even go into apoptosis. Many aging-related diseases are linked to shortened telomeres. Organs deteriorate as more and more of their cells die off or enter cellular senescence.

6 Prof. Dr. H.D.El-Yassin 2013 Lecture 1 Cancer

When normal cells are damaged or old they undergo apoptosis; cancer cells, however, avoid apoptosis. All cancers begin in cells and are caused by mutations. Normally, cells grow and divide to form new cells only when the body needs them. When cells grow old and die, new cells take their place. Mutations can sometimes disrupt this orderly process. New cells form when the body does not need them, and old cells do not die when they should.

Conclusions: 1. DNA synthesis occurs by the process of replication during which, each of the two parental strands of DNA serves as a template for the synthesis of a complementary strand. Thus, each DNA molecule generated by the replication process contains one intact parental strand and one newly synthesized strand. In eukaryotes, DNA replication occurs during the S phase of the cell cycle, which is followed by the G2 phase. The cell divides during the next phase (M), and each daughter cell receives an exact copy of the DNA of the parent cell.

2. In both prokaryotes and eukaryotes, the site at which replication is occurring at any given moment is called the replication fork. As replication proceeds, the two parental strands separate in front of the fork. Behind the fork, each newly synthesized strand of DNA base-pairs with its complementary parental template strand. A complex of proteins is involved in replication. Helicases and unwind the parental strands, and single-strand binding proteins prevent them from reannealing.

3. The major enzyme involved in replication is a DNA polymerase that copies each parental template strand in the 3' to 5' direction, producing new strands in a 5' to 3' direction. Deoxyribonucleoside triphosphates serve as the precursors. One strand of newly synthesized DNA grows continuously, whereas the other strand is synthesized discontinuously in short segments known as Okazaki fragments. These fragments are subsequently joined by DNA ligase.

4. Telomeres. The ends of linear chromosomes are called telomeres. The enzyme telomerase, an RNA-dependant DNA polymerase that carries its own RNA template, is required for their replication.

7 Prof. Dr. H.D.El-Yassin 2013