DNA Replication PDF

DNA Replication Aim of studing -for many anticancer drugs target DNA replication arresting the cancer cell growth. -Many antibiotics inhibit bacterial DNA replication. DNA Replication -Replication of DNA must occur before each cell division. -Replication must be carried out with high fidelity. -DNA...

DNA Replication Aim of studing -for many anticancer drugs target DNA replication arresting the cancer cell growth. -Many antibiotics inhibit bacterial DNA replication. DNA Replication -Replication of DNA must occur before each cell division. -Replication must be carried out with high fidelity. -DNA is duplicated at rates as high as 1000 nucleotides per second. -Replication is based on complementarity. ) Semiconservative( - One strand acts as template (parental DNA)and other is new strand(complementary strand) Phase of DNA Replication Initiation- -Recognition of the position(s) on a DNA molecule where replication will begin -Unwinding of double helix to provide a single stranded template Elongation. - occurring at replication fork where parent DNA is copied. Termination. -Occurs when parent molecule has been completely replicated. -Ligation of newly synthesized DNA segments and reconstitution of chromatin structure. steps of DNA Replication in prokaryotics 1- Unwinding of the 2 DNA strands -Initiation of replication is not random. -starts at special DNA sequences(A-T rich) called replication origin form replication bubble. - replication bubble , the 2 DNA strands separate from each other to serve as DNA templates. -leads to the formation of 2 replication forks and it is bidirectional. -prokaryotic DNA has single origin of replication. So called Theta replication The unwinding occurs by some proteins Dna A protein→melt double stranded DNA at the replication origin Dna B (helicases) → enzymes that unwind the DNA double helix which separate the strands it weakens the hydrogen bonds between the base pairs and pulls the strand apart. Single strand binding (SSB) proteins help helicases by prevent the helix from reforming again Actions of Topoisomerases - unwinding proceeds of double helix in a right handed direction. - relieve torsional strain by break DNA in front of the fork -Topoisomerase I → single strand break, allowing helix to rotate freely -use phosphodiester bond as the swivel point. Topoisomerase II→ Causes a double strand break Anti-Cancer Drugs Against Topoisomerase I is inhibited by→ 1- Topotecan 2- Irinotecan -Topoisomerase II is inhibited by→ 1- Etoposide 2- Teniposide 3- Doxorubicin DNA Gyrases and Topoisomerase IV - DNA Gyrases present in bacteria. similar to type II topoisomerases of eukaryotes. -Bacteria also contain topoisomerase IV. They relieve supercoiling in circular bacterial DNA Gyrases and topoisomerase IV inhibited by→ antibiotics called fluoroquinolones Ex: Ciprofloxacin 2-Primer formation -form from short RNA (7-10 nts) -the sign at which DNA polymerase start to building 3-DNA Synthesis by DNA polymerase (Elongation process) -DNA polymerase synthesizes the new DNA strand starting from primers. - One strand is synthesized continuously. This strand is called leading strand in the 5’ to 3’ direction. - other strand is not synthesized continuously. The lagging strand is synthesized in fragments called “Okazaki fragments”. Multiple primers are synthesized on the lagging strand by the enzyme, primase. - each Okazaki fragment is :200 nts in length in Euk and is synthesized in the 5” to 3” direction -After that RNA primers are removed, and the gaps are filled by DNA ligase. DNA polymerases in prokaryotics DNA polymerase I: main enzyme in DNA repair, it has 3 functions: 5'→3' polymerase activity building DNA strand 5' → 3' exonuclease activity for removal of primer and DNA repair 3' → 5' exonuclease activity for proofreading. DNA polymerase II: not involved in replication  DNA polymerase III: main enzyme in DNA replication 5' → 3' polymerase activity building DNA strand 3' → 5' exonuclease activity for proofreading and high fidelity. (rate of error 1:3x109). - DNA Synthesis at end of chromosome cannot replicate in linear DNA molecule (chromosomal DNA). Because at 3’ end of parental strand primer cannot be synthesized. thus daughter one will be shorter. - telomere consists of 1000 or repeated TG- rich sequences at the ends of linear DNA. - telomerase recognizes the telomere region and elongates in the 5’ to 3’ direction. -Telomerase is a ribonucleoprotein, a complex of protein and RNA. (451 nts in human ) What Happens if Telomerase Activity is Low? - 3’ end of the parental strand cannot be copied and will be shorted by 100-200 nts NB: activity decrease by age so lead to cell aging Processivity :measure of the average number of nucleotides added by a DNA polymerase enzyme Proofreadin: Identifies copying errors and corrects them. Eukaryotic Prokaryotic Multiple origin of replication(30000) Single Origin DNA polymerase 5 Types: 3 types: 1- α polymerase for synthesis of primer. 1- Type I main in DNA repair. 2- β polymerase for DNA repair and gap filling 2- Type II. 3- γ polymerase for replication of mt DNA 3- Type III main in replication 4- δ polymerase for elongation of leading strand. 5- € polymerase for elongation of lagging strand Okazaki fragment: shorter (100- 200bp) longer (1000 – 2000 bp) The DNA is linear with telomeres and need telomerase enz Circular, not need telomerase More complex, more slower(100 nuc/sec) Less complex, faster(1000 nuc/sec. Protein Function. DnaA protein Melt the double stranded DNA at the origin of replication Helicases Unwinding of DNA forming a single strand. Single stranded Keep the 2 separated DNA strands apart and prevent them binding proteins from rewinding and protects single stranded DNA from nucleases. Topoisomerase -Release the DNA supercoiling and linearize DNA strands -also responsible for the rewindining of DNA. Primase Synthesizing the RNA primers. DNA poly. III DNA synthesis. DNA poly. I Erase the RNA primer and fill the gaps lifted. DNA ligase Join the Okazaki fragments together on lagging strand DNA damage and repair DNA damage 1-Ionizing radiation such as gamma rays and X-rays. 2-Ultraviolet rays, especially the UV-C rays (~260 nM) 3-Reactive oxygen species produced during cellular metabolism 4-Chemicals in the environment, e.g., hydrocarbons in cigarette ,microbial products such as aflatoxins 5-Chemicals used in chemotherapy Depurination of DNA: - change in purine which removed from deoxyribose sugar by hydrolysis of N-glycosidic link Deamination -Cytosine deamination→ uracil. -Adenine →hypoxanthine. -Guanine d→ xanthine.