5. Finishing Replication.pdf

Molecular Biology and Cytogenetics 5. Finishing Replication Instructor: Dr. Mohammad Abukhalil Finishing replication in Prokaryotes ⚫ Termination of DNA replication in bacteria is completed when two replication forks meet at the terminus of the chromosome. ⚫ When a circular c...

Molecular Biology and Cytogenetics 5. Finishing Replication Instructor: Dr. Mohammad Abukhalil Finishing replication in Prokaryotes ⚫ Termination of DNA replication in bacteria is completed when two replication forks meet at the terminus of the chromosome. ⚫ When a circular chromosome finishes replication, the two new circles may be physically interlocked or catenated. ⚫ Decatenation of interlocked circles is carried out by topoisomerase type II by breaking the phosphodiester bonds. The end replication problem ⚫ Each DNA replication, end of DNA loses 50~100 nt. ⚫ Lose genes near the end of chromosome I- Attach a protein to 5’ end to serve as primer ⚫ Some bacteria have linear chromosome and animal virus. (Note: most bacteria have circular chromosome to solve end problem) ⚫ They have a terminal protein attached covalently to their 5′ DNA ends that provide an OH that replace the 3’OH normally provided by an RNA primer. II- Telomerase ⚫ Is a ribonucleoprotein ⚫ Responsible for elongating the 3` strand Telomere ⚫ Repetitive regions at the very ends of chromosomes are called telomeres, and they're found in a wide range of eukaryotic species, from human beings to unicellular protists. ⚫ Telomeres act as caps that protect the internal regions of the chromosomes, and they protect the end of the chromosome from fusion with neighboring chromosomes. ⚫ Telomeres consist of multiple tandem repeats (from 20 to several hundred) of a short sequence, usually of six bases (TTAGGG, in vertebrates including humans).  Telomerase 1. RNA component: Template. 2. Protein component: reverse transcriptase enzyme. Telomerase ⚫ Telomerase carries a small segment of RNA, complementary to the six-base-pair telomere repeat. ⚫ The enzymatic portion of telomerase resembles other reverse transcriptases, proteins that synthesize DNA using an RNA template. ⚫ After telomerase has elongated the 3’-ends, the complementary strand can be filled in by normal RNA priming followed by elongation by DNA polymerase and joining by ligase. Regulation of telomerase activity ⚫ Telomere length regulation involves the accessibility of telomeres to telomerase. ⚫ Length control involves a number of factors including: ⚫ Proteins POT1, TRF1, and TRF2 ⚫ t-loop formation ⚫ A telomere-specific protein complex forms called shelterin. When the telomere is long enough: ⚫ POT1 levels are high at the 3′ overhang. ⚫ The action of telomerase is blocked. When the telomere is too short: ⚫ Little or no POT1 is present at the 3′ end. ⚫ Telomerase is no longer inhibited. A model for t-loop formation ⚫ The 3′ single-stranded DNA tail invades the double- stranded telomeric DNA. ⚫ A loop forms in which the 3′ overhang is base paired to the C strand sequence. ⚫ The t-loop may aid in preventing telomerase access. Regulation of telomerase activity ⚫ NO telomerase → NO maintenance of telomere → telomere loss (50-150 bp/end/division) → cell apoptosis ⚫ Telomerase → telomere maintenance → immortal cell Telomerase is tightly regulated  Telomerase, aging, and cancer ⚫ In most human somatic cells, not enough telomerase is expressed to maintain a constant telomere length: Progressive shortening of telomeres. ⚫ High levels of telomerase activity in ovaries, testes, rapidly dividing somatic cells, and cancer cells. ⚫ Some stem cells, notably those in tissues that must be replenished at a high rate throughout life—bone marrow or gut lining, for example— retain full telomerase activity. Telomerase and aging: the Hayflick limit ⚫ The Hayflick limit is the point at which cultured cells stop dividing and enter an irreversible state of cellular aging (senescence). ⚫ Proposed to be a consequence of telomere shortening. Dyskeratosis congenita: loss of telomerase activity ⚫ Premature aging syndrome. ⚫ Problems in tissues where cells multiply rapidly and where telomerase is normally expressed. ⚫ Two forms of dyskeratosis congenita: ⚫ X-linked recessive ⚫ Autosomal dominant X-linked recessive dyskeratosis congenita ⚫ Mutations in dyskerin gene. ⚫ Dyskerin is a pseudouridine synthase that binds to small nucleolar RNAs and to telomerase RNA. ⚫ Patients with dyskerin mutations have 5-fold less telomerase activity than unaffected siblings. Autosomal dominant dyskeratosis congenita ⚫ Mutations in telomerase RNA gene in the pseudoknot domain. ⚫ Partial loss of function of telomerase RNA. Gene therapy for liver cirrhosis ⚫ Inhibition of liver cirrhosis in mice by telomerase gene delivery. ⚫ Why hasn’t this gene therapy strategy progressed to human trials?