BMB 251 1st Edition Lecture 15 Outline of Last Lecture I. DNA replicationa. Leading Strandb. Lagging StrandII. DNA primaseIII. RNA primersIV. RNAse HV. DNA ligaseVI. DNA helicase VII. DNA polymeraseVIII. SSB proteinsIX. Sliding clampX. DNA topoisomerases Outline of Current Lecture XI. ClickersXII. DNA replication originsXIII. Replication unitsXIV. Thymidine analog bromodeoxyuridine (BrdU)XV. Origin Recognition ComplexXVI. End Replication problemsXVII. Replicative cell senescenceCurrent Lecture- Clicker Question 1: Within each active replication bubble, there are how many DNA polymerase molecules o 4- Clicker Question 2: Within each active replication bubble, there are how many helicase molecules?o 2- DNA: normally very stable; H-bonds between bases of two strands - DNA replication is begun by special initiator proteins which bind to double-stranded DNA and pry two strands apart, breaking the H-bonds- Replication origins: positions at which DNA is first openedo A-T held together by two H-bonds versus three H-bonds of G-C; A-T is therefore easier topull apart  regions of DNA enriched in A-T pairs = typically found at replication origins- The only part of replication bacteria can control is initiation  highly regulated These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o Initiator proteins bind in multiple copies to specific sites of replication origin, wrapping DNA around proteins to form a complexo This complex attracts helicase bound to helix loader (analogous to clamp loader) o Helicase unwinds DNA, exposing enough for primase to synthesize RNA primer that begins the loading strando **Interaction of initiator protein with replication origin = carefully regulated, with initiation only occurring when sufficient nutrients are available- Autoradiography: reveals pattern of radioactive DNA via development of photographic emulsion o Rates and direction of replication fork movement can be determined from this- Replication origins tend to be activated in clusters (aka replication units); each unit containing between 20-80 origins- New replication units seem to be activated at different times during cell cycle until all of DNA is replicated- Within replication unit, each origin is spaced out at intervals of 30,000-250,000 nucleotides- Like bacteria, replication forks are formed in pairs/create replication bubbles- DNA replication occurs during DNA synthesis (S) phase of cell cycle, which takes about eight hours to complete in mammals- Replication origins are not all activated simultaneously and DNA in each replication unit is replicated during only a small part of total S-phase interval- Thymidine analog bromodeoxyuridine (BrdU): labels newly synthesized DNA o DNA with BrdU within it during M-phase can be recognized by altered staining properties or by means of BrdU antibodies- Replication forks only stop when coming into contact with another replication fork/end of chromosome- In bacteria, DNA sequence that serves as origin of replication needs:o Binding site for initiator protein called ORC (origin recognition complex)o Stretch of DNA rich in A and T  easy to unwindo At least one binding site for proteins to help attract ORC to the origin DNA- Proteins that bind to ORC-origin to regulate origin activity: helicase and helicase loading proteinsCdc6 and Csd1- Passage of G1  S-phase is triggered by activation of protein kinases (Cdks) that lead to dissociation of helicase loading proteins, activation of helicase, unwinding of origin DNA and loading of remaining replication proteins, including DNA polymerase- Each origin of replication can fire once and only once in a cell cycle- ORC protein seems to be less specific in humans than in yeast  chromatin structure, rather than DNA sequences, define their origins of replication- Eukaryotic chromosomes composed of equal amounts of DNA and proteino Cell must not only replicate DNA but synthesize new proteins as well- Histones are synthesized mainly in the S-phase- To replicate chromosomes, chromatin-remodeling proteins, which destabilize the DNA-histone interphase, are required- As replication fork passes through chromatin, most of old histones remain DNA-bound and are distributed to daughter strands following the forko Need just as many new histones as old ones following replicationo **Histone chaperones: orderly and rapid addition of new H3-H4 tetramers and H2A-H2Bdimers h=bind fork require this, which bind highly basic histones and release them for assembly only in the appropriate context- Histones are subject to many covalent modificationso Duplication of these modifications  epigenetic inheritance where a heritable change incell’s phenotype occurs without a change in the nucleotide sequence- “End-replication problems”: no place to produce the RNA primer needed to start last Okazaki fragment at very tip of linear DNA once fork reaches end of DNA o Eukaryotes have specialized nucleotide sequences at ends of chromosomes that are incorporated into structures called telomereso Repeated nucleotide sequences; recognized by sequences-specific DNA binding proteins which attract an enzyme called telomerase (replenishes repeating sequences every time cell divides)o Use “reverse transcriptases” to synthesize DNA from RNA template and elongate telomeres in the 5’ to 3’ directiono T-loop: extra length of single-stranded 3’ end of telomere loops back to creates a terminus into DNA telomereo **T-loop protects ends of chromosomes from degradative enzymes and clearly distinguishes them from ends of broken DNA molecules in need of repair - Somatic cells are born with full complement of telomerase repeatso Stem cells (ex. In tissues such as none marrow/skin), male sperm cells and immune cells retain full telomerase activityo Other cells have lower levels telomerase is turned down so it cant keep up with chromosome replication  cells lost around 100-200 nucleotides from each telomere with every cell division- Replicative cell senescence: after many cell generations, descendants inherit defective chromosomes without fully replicated tips, which then withdraw permanently from cell cycle and stop dividingo **Important aspect of aging and replication senescence makes cell less likely to get cancer (controversial idea)- Dyskeratosis congenital: organism carries one functional and one nonfunctional copy of telomerase RNA