DNA replication.pdf

Molecular Genetics DNA REPLICATION Molecular Biology DNA Replication Necessary during cell division to ensure the formation of two identical daughter cells DNA replication only occurs during S-phase DNA replication is semi-conservative The two strands are separated and used as a template for the for...

Molecular Genetics DNA REPLICATION Molecular Biology DNA Replication Necessary during cell division to ensure the formation of two identical daughter cells DNA replication only occurs during S-phase DNA replication is semi-conservative The two strands are separated and used as a template for the formation of the new strand. The final result is that each new DNA has one ‘old’ strand and one ‘new’ strand DNA replication is bi-directional DNA replication start at “origin of replication” points and continues in both directions, creating a replication bubble. Mammalian genomes have thousands of origins of replications that create replication bubbles that end up fusing to each other DNA replication is bi-directional Pre-replication Complex Origins of replication are recognized by ORC proteins and MCM proteins that form the pre-Replication complex. Their function is to recruit the DNA replication machinery during S-phase, and thus, the pre-replication is heavily regulated during the cell cycle The Key to understand DNA replication: the enzymatic activity of DNA polymerase DNA synthesis always proceeds in the 5′→3′ direction because chain growth results from formation of a phosphodiester bond between the 3′ oxygen of a growing strand and the α phosphate of a dNTP In contrast, DNA polymerases cannot initiate chain synthesis de novo: they require a short, preexisting RNA or DNA strand, called a primer, to begin chain growth and a template. With a primer base-paired to the template strand, a DNA polymerase adds deoxynucleotides to the free hydroxyl group at the 3′ end of the primer as directed by the sequence of the template strand: DNA replication is semi-discontinuous Two strands: one direction, two orientations Leading strand: continuous replication Lagging strand: discountinuous through formation of Okazaki fragments Enzymatic activities required for DNA replication Helicases: unwinding of the parental DNA strands Primases: To form the RNA primers required for DNA plolymerases Polymerases: to polymerize nucleotides using the ‘old strand’ as a template’ Topoisomerases: to relieve tensions created by the replication bubble Ligases: To ligate the different strands that are being formed (especially in the lagging strands) Molecular Biology Molecular biology Helicase to unwind the parental DNA strands. Requires ATP. RPA a trimeric complex that binds the single strand DNA (ssDNA) separated by the helicase. Its function is to protect ssDNA from nucleases and to prevent the formation of secondary structures Leading strand The leading strand is synthesized by a complex of: DNA polymerase ε (Pol ε) PCNA: trimeric complex that threads onto DNA and prevents the whole complex from falling off the template. It also displaces primase at the beginning of replication. Known as the sliding clamp Rfc (Replication factors): Helps to stabilize the PCNA-Polε complex during replication. It also opens the PCNA ring to load onto DNA, thus it is also known as the clamp loader Molecular biology Primase: a complex formed by: RNA primase: synthesizes short (~10nt) RNA primer DNA polymerase-alpha: adds ~20nt DNA to the RNA primer Primase makes one primer for the leading strand and multiple primers for the lagging strand (one for each Okazaki fragment) Lagging Strand The lagging strand is synthesized by a complex of: DNA polymerase δ (Pol δ)-PCNA-Rfc Function: Polymerize the lagging strand Removes RPA and primases following primer synthesis Other functions required: Ribonucleases to remove the RNA primer from the newly synthesized strand DNA ligases to join Okazaki fragments together DNA replication is concurrent Concurrent means that both the lagging and leading strand are synthesized at the same time in both replication forks DNA replication is concurrent Video Telomeres The natural ends of chromosomes Composed of a highly repetitive sequence Human telomeres, for example, range in size from 4-12 kilobases and consist of approximately 300-8,000 precise repeats of the sequence CCCTAA/TTAGGG Telomere end in a G-Tail A common feature of all telomeres is the orientation of the Grich strand. This strand makes up the 3'-end of the chromosome, and the terminal portion of the G-rich strand is single-stranded, generating a so-called "G-tail." The actual length of the G-tail is somewhat variable, averaging between 75-300 nucleotides in humans. Telomeres form loops Most eukaryotic chromosomes bend back on themselves to form a physical loop at the telomere. This is caused by: -The nature of the G-tail , which can complement internal tracks of telomere sequence -The presence of Telomere specific proteins that recognize the telomere sequence, bind telomeres and assist in the formation of the loop Human Telomeres 4-12Kb SHELTERING: Telomeric Proteins Telomeres are recognized by a series of proteins that can be classified as : -Telomere Specific proteins: Example: TRF1 and TRF2 -Telomere non specific proteins: Example: Ku The main function of telomeres is protection Exonucleases DNA repair Recombination Telomeres main function is protection Telomere replication problem Telomeres get shorter every replication Telomeres get shorter every replication Telomeres shortening leads to unprotected ends Telomere shortening leads to senescence Short telomeres play a central role in the development of age-related diseases DISEASE LEVEL CNS (CENTRAL NERVOUS SYSTEM) DISEASES ALZHEIMER’S, PARKINSONS, DEMENTIA DIABETES ORGAN LEVEL BRAIN CELLULAR Senescence & Apoptosis CARDIOVASCULAR DISEASE, HYPERTENSION & ATHEROSCLEROSIS Short Telomeres RETINA INFERTILITY, MENOPAUSE & «ANDROPAUSE» REPRODUCTIVE ORGANS DNA Damage MACULAR DEGENERATION DNA Damage Senescence & Apoptosis WRINKLES, ARTHRITIS, OSTEOPOROSIS STEM CELLS WEAK INMUNITY / ASTHMA / ALLERGIES CANCER Source: Recharge Biomedical Clinic & DR. Ed Park © 2011 Life Length. All rights reserved. Reproduction of this document or any portion thereof without prior written consent is prohibited. Page 1 Telomerase can extend Telomere ends Telomerase can extend Telomere ends Sheltering recruits Telomerase to telomeres