Molecular Biology Lv2 DNA Replication Lecture 4 PDF

DNA REPLICATION A. GENERAL FEATURES OF CHROMOSOMAL REPLICATION ► 1. DNA replication is semiconservative  DNA REPLICATION A. GENERAL FEATURES OF CHROMOSOMAL REPLICATION  2. Most DNA replication is bidirectional DNA replication proc...

DNA REPLICATION A. GENERAL FEATURES OF CHROMOSOMAL REPLICATION ► 1. DNA replication is semiconservative  DNA REPLICATION A. GENERAL FEATURES OF CHROMOSOMAL REPLICATION  2. Most DNA replication is bidirectional DNA replication proceeds bidirectionally from a given starting site, with both strands being copied at each fork. Thus two growing forks emerge from a single origin site.  DNA REPLICATION A. GENERAL FEATURES OF CHROMOSOMAL REPLICATION  Circular DNA molecules present in bacteria, plasmids, and some viruses have one origin  In E. coli - about 42 minutes to replicate the single circular chromosome of 4,639,221 bp.  Replication speed - is about 1000 bp per second per fork.  DNA REPLICATION A. GENERAL FEATURES OF CHROMOSOMAL REPLICATION  4.1. E. coli Replication Origin The E. coli replication origin oriC is an ≈240-bp DNA segment present at the start site for replication. Legend to Figure. Consensus sequence of the minimal bacterial replication origin based on analyses of genomes from six bacterial species. Similar sequences constitute each origin; the 13-bp repetitive sequences (orange) are rich in adenine and thymine residues. The 9-bp repetitive sequences (brown) exist in both orientations; that is, the lower-right sequence, read right to left, is the same as that of the upper-left sequence, read left to right. These sequences are referred to as 13-mers and 9-mers, respectively. Indicated nucleotide position numbers are arbitrary.  DNA REPLICATION A. GENERAL FEATURES OF CHROMOSOMAL REPLICATION Legend to Figure. DNA replication and cell division in a prokaryote. (a) In a bacterial cell, the partially replicated circular chromosome (blue) is attached to the plasma membrane at the origins of the two daughter DNAs (step 1 ). The origins of the replicated chromosomes have independent points of attachment to the membrane and thus move farther apart as new membrane and cell wall forms midway along the length of the cell (step 2). Continued formation of more sections of membrane and cell wall gives rise to a septum dividing the cell (step 3), leading to division of the cytoplasm into two daughter cells, each with a chromosome attached to the plasma membrane (step 4).  DNA REPLICATION A. GENERAL FEATURES OF CHROMOSOMAL REPLICATION 5. Three Common Features of Replication Origins  First, replication origins are unique DNA segments that contain multiple short repeated sequences.  Second, these short repeat units are recognized by multimeric origin-binding proteins. These proteins play a key role in assembling DNA polymerases and other replication enzymes at the sites where replication begins.  And third, origin regions usually contain an AT-rich stretch. This property facilitates unwinding of duplex DNA because less energy is required to melt A·T base pairs than G·C base pairs.  DNA REPLICATION B. THE DNA REPLICATION MACHINERY Problems in the copying of DNA by DNA polymerases:  DNA polymerases are unable to melt duplex DNA (i.e., break the interchain hydrogen bonds) in order to separate the two strands that are to be copied.  All DNA polymerases so far discovered can only elongate a preexisting DNA or RNA strand, the primer; they cannot initiate chains.  The two strands in the DNA duplex are opposite (5′ → 3′ and 3′ → 5′) in chemical polarity, but all DNA polymerases catalyze nucleotide addition at the 3′-hydroxyl end of a growing chain, so strands can grow only in the 5′ → 3′ direction.  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  1. DnaA Protein Initiates Replication in E. coli Legend to Figure. Model of initiation of replication at E. coli oriC. The 9-mers and 13-mers are the repetitive sequences shown in Figure 4. Multiple copies of DnaA protein bind to the 9-mers at the origin and then “melt” (separate the strands of) the 13-mer segments. The sole function of DnaC is to deliver DnaB, which is composed of six identical subunits, to the template. One DnaB hexamer clamps around each single strand of DNA at oriC, forming the prepriming complex. DnaB is a helicase, and the two molecules then proceed to unwind the DNA in opposite directions away from the origin.  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  2. DnaB Is an E. coli Helicase That Melts Duplex DNA 3 Rep-protein ’ 5’ 3’ SSB proteins DnaB 5 ’  DnaB and Rep-protein  SSB proteins  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  3. E. coli Primase Catalyzes Formation of RNA Primers for DNA Synthesis  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  4. At a Growing Fork One Strand Is Synthesized Discontinuously from Multiple Primers Main enzymes: DNA polymerase III (Pol III) DNA polymerase I DNA ligase  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  DNA ligase  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  5. E. coli DNA Polymerase III Catalyzes Nucleotide Addition at the Growing Fork  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  The DNA polymerase III holoenzyme: 10 different polypeptides.  The core polymerase is composed of three subunits: - a subunit contains the active site for nucleotide addition - e subunit is a 3’  5’ exonuclease that removes incorrectly added (mispaired) nucleotides from the end of the growing chain : “proofreading” activity -  subunit is not known.  The central role of the remaining subunits is to convert the core polymerase from a distributive enzyme, which falls off the template strand after forming relatively short stretches of DNA containing 10 – 50 nucleotides, to a processive enzyme, which can form stretches of DNA containing up to 5 × 105 nucleotides without being released from the template. The key to the processive nature of DNA polymerase III is the ability of the b subunit to form a donut-shaped dimer around duplex DNA and then associate with and hold the catalytic core polymerase near the 3’ terminus of the growing strand.  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  6. The Leading and Lagging Strands Are Synthesized Concurrently  DNA REPLICATION B. THE DNA REPLICATION MACHINERY  8. Telomerase prevents progressive shortening of lagging strands during eukaryotic DNA replication Legend to Figure. Mechanism of action of telomerase. This ribonucleoprotein complex elongates the 3’ telomeric end of the lagging-strand DNA template by a reiterative reverse transcription mechanism. The action of the telomerase from Oxytricha, which adds a T4G4 repeat unit, is depicted. The telomerase contains an RNA template (red) that base-pairs to the 3’ end of the lagging-strand template. The telomerase catalytic site (green) then adds deoxyribonucleotides (blue) using the RNA molecule as a template; this reverse transcription proceeds to position 35 of the RNA template (step 1). The strands of the resulting DNA-RNA duplex are then thought to slip relative to one another, leading to displacement of a single-stranded region of the telomeric DNA strand and to uncovering of part of the RNA template sequence (step 2). The lagging-strand telomeric sequence is again extended to position 35 by telomerase, and the DNA-RNA duplex undergoes translocation and hybridization as before (steps 3 and 4). The slippage mechanism is thought to be facilitated by the unusual base pairing (black dots) between the displaced G residues, which is less stable than Watson-Crick base pairing. Telomerases can add very long stretches of repeats by repetition of steps (4) and (5).  DNA REPLICATION C. THE ROLE OF TOPOISOMERASES IN DNA REPLICATION  1. Type I Topoisomerases Relax DNA by Nicking and Closing One Strand of Duplex DNA  DNA REPLICATION C. THE ROLE OF TOPOISOMERASES IN DNA REPLICATION  2. Type II Topoisomerases Change DNA Topology by Breaking and Rejoining Double- Stranded DNA DNA gyrase: functions to introduce negative supercoils at or near the oriC site in the DNA template; as DnaA can initiate replication only on a negatively supercoiled template removes the positive supercoils that form ahead of the growing fork during elongation of the growing strands  DNA REPLICATION C. THE ROLE OF TOPOISOMERASES IN DNA REPLICATION  3. Replicated Circular DNA Molecules Are Separated by Type II Topoisomerases  4. Linear Daughter Chromatids Also Are Separated by Type II Topoisomerases Legend to Figure. Completion of replication of circular DNA molecules. Denaturation of the unreplicated terminus followed by supercoiling overcomes the steric and topological constraints of copying the terminus. At least with SV40 DNA, the final two steps (synthesis and decatenation) can occur in either order depending on experimental conditions. Parental strands are in dark colors; daughter strands in light colors.