DNA Replication PDF

REPLICATION OF DNA DNA replication is the process by which two identical replicas of a DNA are obtained from the original DNA strand. The DNA replicates before the cell division occurs. Both prokaryotic and eukaryotic DNA replicate in a semi-conservative manner. However, there are a few differences...

REPLICATION OF DNA DNA replication is the process by which two identical replicas of a DNA are obtained from the original DNA strand. The DNA replicates before the cell division occurs. Both prokaryotic and eukaryotic DNA replicate in a semi-conservative manner. However, there are a few differences between the prokaryotic and eukaryotic DNA replication based on their size and complexity in genetic material. Each strand of the DNA double helix has all the information needed to reconstruct the other half by the mechanism of base pairing. In most prokaryotes, DNA replication begins at a single point and continues in two directions. In eukaryotic chromosomes, DNA replication occurs at hundreds of places. Replication proceeds in both directions until each chromosome is completely copied. The sites where separation and replication occur are called replication forks. Duplicating DNA Before a cell divides, it duplicates its DNA in a process called replication. Replication ensures that each resulting cell will have a complete set of DNA. DNA Template During DNA replication, the DNA molecule separates into two strands, then produces two new complementary strands following the rules of base pairing. Each strand of the double helix of DNA serves as a template for the new strand. Eukaryotic DNA Replication Prokaryotic DNA Replication The eukaryotic DNA is present inside This is the process by which the genome of the nucleus. The process involves prokaryotic cells duplicates so that it can be three steps – initiation, elongation transformed into a daughter cell. The DNA and termination. DNA helicase and is circular, double-stranded and found in the single-strand binding proteins are cytoplasm. A single origin of replication responsible for unwinding and results in the formation of two replication stabilization. The replication process forks. is halted as the leading strand of one The initiation and elongation of prokaryotic replication bubble meets the lagging DNA is carried out by the enzyme DNA strand of another replication bubble. polymerase III. The nucleotides are added in the 5’ to 3’ direction. The nicks formed are joined by the enzyme ligase. How Replication Occurs DNA replication is carried out by enzymes that “unzip” a molecule of DNA. Helicase breaks hydrogen bonds between base pairs and the two strands of DNA unwind. How does DNA replicate? 1)UNWIND: Topoisomerase unwinds the coiled strands of DNA. 2) UNZIP: DNA Helicase “unzips” the strands of DNA breaking the hydrogen bonds, creating two template (parent) strands for replication 3) HOLD OPEN: Single-Strand Binding proteins (SSBs) keep strands separated 4) BASE PAIRING: DNA polymerase III bonds free nucleotides with nucleotides on each template (parent) strand using base pairing rules 5) PROOFREAD: DNA Polymerase I proofreads new strands and backtracks to correct errors 6) JOINING NUCLEOTIDES: DNA ligase bonds backbone together. Model of DNA Replication DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond Model of DNA replication Model of DNA replication Concepts and terms to understand: Why are gyrase and helicase required? The difference between a template and a primer? The difference between primase and polymerase? What is a replication fork and how many are there? Why are single-stranded binding (SSB) proteins required? How does synthesis differ on leading strand and lagging strand? Which is continuous and semi-discontinuous? What are Okazaki fragments? Replication of circular DNA in E. coli: 1. Two replication forks result in a theta-like () structure. 2. As strands separate, positive supercoils form elsewhere in the molecule. 3. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate. Rolling circle model of DNA replication: 1. Common in several bacteriophages including . 2. Begins with a nick at the origin of replication. 3. 5’ end of the molecule is displaced and acts as primer for DNA synthesis. 4. Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly). 5. During viral assembly the DNA is cut into individual viral chromosomes. DNA replication in eukaryotes: Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges: Major checkpoints in the system 1. Cells must be large enough, and the environment favorable. 2. Cell will not enter the mitotic phase unless all the DNA has replicated. 3. Chromosomes also must be attached to the mitotic spindle for mitosis to complete. 4. Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks).  Each eukaryotic chromosome is one linear DNA double helix Average ~108 base pairs long With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days. Solution ---> DNA replication initiates at many different sites simultaneously. Rates are cell specific! Replication forks visible in Drosophila What about the ends (or telomeres) of linear chromosomes? DNA polymerase/ligase cannot fill gap at end of chromosome after RNA primer is removed. If this gap is not filled, chromosomes would become shorter each round of replication! Solution: 1. Eukaryotes have tandemly repeated sequences at the ends of their chromosomes. 2. Telomerase (composed of protein and RNA complementary to the telomere repeat) binds to the terminal telomere repeat and catalyzes the addition of of new repeats. 3. Compensates by lengthening the chromosome. 4. Absence or mutation of telomerase activity results in chromosome shortening and limited cell division. Synthesis of telomeric DNA by telomerase  Prokaryotic Replication vs Eukaryotic Replication Following are the important difference between prokaryotic and eukaryotic replication: Prokaryotic Replication Eukaryotic Replication This process occurs in prokaryotic cells. This process occurs in eukaryotic cells. It is a continuous process. This process occurs in the S-phase of cell cycle. Circular, double-stranded DNA Linear, double-stranded DNA with end The DNA replicates in the cytoplasm The DNA replicates in the nucleus Single origin of replication Multiple origins of replication Small amount of DNA The DNA is 50 times more than prokaryotic DNA DNA polymerase I and III are involved DNA polymerase ɑ, δ and ε are involved. Large okazaki fragments Small okazaki fragments The process is rapid, 2000 base pairs per second The process is slow, 100 base pairs per second Two circular chromosomes are obtained Two sister chromatids are obtained DNA gyrase is required DNA gyrase is not required Final Step - Assembly into Nucleosomes: As DNA unwinds, nucleosomes must disassemble. Histones and the associated chromatin proteins must be duplicated by new protein synthesis. Newly replicated DNA is assembled into nucleosomes almost immediately. Histone chaperone proteins control the assembly.