MCBL121 Lecture 4: Genomes and Replication PDF

BIOL/MCBL 121 INTRODUCTORY MICROBIOLOGY Lecture 4 Genomes and Replication Copyright Ansel Hsiao 2021 Objectives • Describe process of chromosomal and plasmid replication in bacteria • Describe evolutionary strategies for plasmid maintenance Copyright Ansel Hsiao 2021 Bacterial DNA Replication...

BIOL/MCBL 121 INTRODUCTORY MICROBIOLOGY Lecture 4 Genomes and Replication Copyright Ansel Hsiao 2021 Objectives • Describe process of chromosomal and plasmid replication in bacteria • Describe evolutionary strategies for plasmid maintenance Copyright Ansel Hsiao 2021 Bacterial DNA Replication • Nucleoid is replicated via replisome complexes, beginning at the origin of replication • Replication of cellular DNA in most cases is semiconservative • Each daughter cell receives one parental and one newly synthesized strand Copyright Ansel Hsiao 2021 Replisome • DNA helicase – unwinds the two strands of DNA • SSBs (single-stranded DNA binding proteins) – keep the single DNA strands apart during DNA synthesis • Clamp loader and sliding clamps: recruit DNA polymerase III and tether it to the DNA • DNA polymerase III – synthesizes new DNA strands • DNA primase – produces RNA primers Copyright Ansel Hsiao 2021 RNA primers are required for DNA replication • DNA primase is an RNA polymerase • The primase synthesizes small RNA molecules (10 -12 nucleotides in length) that act as primers (providing the 3’-OH group) for DNA polymerase III to synthesize new DNA strands • Evolutionary remnant: RNA was the first genetic material Copyright Ansel Hsiao 2021 Replication Forks • Leading strand: steady growth following the DNA helicase. • Lagging strand: Okazaki fragments • DNA primase is loaded to new sites as the replication fork moves • DNA Polymerase III synthesizes ~ 1 Kb DNA pieces following each RNA primer Leading Strand Copyright Ansel Hsiao 2021 Copyright Ansel Hsiao 2021 Completion of DNA replication • RNase H removes the RNA primers (recognizes DNA/RNA heteroduplexes and removes the RNA) • One primer for each leading strand • Many primers on the lagging strand – one per Okazaki fragment • DNA polymerase I fills the gaps in the DNA strand • DNA ligase seals the junctions Copyright Ansel Hsiao 2021 Termination of Replication • Replication ends at defined termination (ter) sites located opposite to the origin • Topoisomerase IV catalyzes a breaking and re-joining event that passes the chromosomes through one another Copyright Ansel Hsiao 2021 Plasmids • Extrachromosomal DNA, usually circular • Size varies: several kilobase (Kb) to megabase (Mb) • Copy numbers varies: 1 to several hundred per cell Copyright Ansel Hsiao 2021 Plasmids • Has its own origin of replication • A plasmid is a replicon: depends on chromosomal genes, such as DNA polymerases etc • Plasmid origins can be stringent or relaxed: • Stringent: Plasmid replicates only with chromosome • Relaxed: Plasmid replication is independent of chromosomal replication Copyright Ansel Hsiao 2021 Plasmids • Important for adaptation to changing environments (e.g. pathogenesis, antibiotic resistance) • Plasmid curing: loss of plasmid if it no longer confers a benefit • Selective pressures: availability of certain nutrients or antibiotics • Can be moved between cells via transformation or conjugation Copyright Ansel Hsiao 2021 Plasmids • Plasmids have evolved tricks to ensure their inheritance: • High-copy-number plasmids segregate randomly to daughter cells – many copies increase chances of each daughter cell inheriting some • Plasmids can carry genes that confer benefits on cells with them (e.g. drug resistance, ability to cause disease) • Low-copy-number plasmids: • • Lower copy number reduces metabolic demands on the cell, and increases fitness of cells making plasmids Segregation systems Copyright Ansel Hsiao 2021 Plasmid Segregation – ParMRC system • parC: sequences on the plasmid that bind to ParR • ParR: a DNA-binding protein that binds to plasmid • ParM: an actin-like protein that forms filaments • ParM filaments as they polymerize push plasmids to opposite ends of the dividing cell Copyright Ansel Hsiao 2021 Plasmid Addiction • A toxin-antitoxin module in plasmids for self-maintenance • Pressure on cells to maintain plasmids • Two adjacent genes on the plasmid encoding a toxin (protein) and an antitoxin (RNA) • RNA is not as stable as the toxin – need to be constantly produced • Cells need to maintain plasmid to have constant source of antitoxin against the plasmidexpressed toxin Copyright Ansel Hsiao 2021 Plasmid Replication • Bidirectional replication • Similar to chromosomal replication • Unidirectional (“rolling circle”) replication • Starts at a nick on a single DNA strand • Provides 3′-OH for synthesizing a new strand (plus strand) • The old plus strand is released and used as the template to synthesize the new minus strand (no Okazaki fragments) Copyright Ansel Hsiao 2021 Rolling circle replication Single stand nick at oriTOrigin of transfer Copyright Ansel Hsiao 2021