Bacterial DNA replication, plasmids, chromosomes, archaea, and PCR

Flashcards covering key concepts from the lecture notes on bacterial DNA structure, replication initiation/elongation/termination, plasmids, genome examples, archaeal replication, and PCR.

What is the bacterial origin of replication called and which protein initiates replication by binding there?

oriC; initiator protein DnaA binds to oriC to start replication.

Which helicase unwinds DNA at the origin in bacteria?

DnaB helicase (DNA B).

Which protein loader delivers the helicase to the origin during initiation?

DnaC (the helicase loader).

What is the primosome in bacterial DNA replication?

The complex of DnaB helicase and DnaG primase that lays down RNA primers for replication.

What is the main replicative DNA polymerase in E. coli?

DNA polymerase III (POL3).

Which enzyme is primarily responsible for primer removal and maturation on the lagging strand?

DNA polymerase I (POL1).

Which enzyme seals remaining nicks after maturation?

DNA ligase.

Why are Okazaki fragments formed on the lagging strand?

Because the lagging strand is synthesized discontinuously in the 5' to 3' direction on a 3' to 5' template, creating short fragments later joined.

What is the function of the tau (τ) subunit in the bacterial replisome?

Links the two DNA polymerases and coordinates lagging-strand synthesis via the clamp loader; stabilizes the replisome.

What is the role of the sliding clamp in bacterial DNA replication?

Keeps DNA polymerase attached to DNA, increasing processivity (beta clamp).

What is the function of the clamp loader in replication?

Loads the sliding clamp onto DNA, enabling polymerase attachment; energy-dependent.

What enzymes relieve and introduce supercoils during replication and what are the classes?

Topoisomerases; Class I (topoisomerase I/III) nick one strand to relieve torsion; Class II (topo II, topo IV, DNA gyrase) nick both strands and can generate/relax supercoils.

What is negative supercoiling and which predominates in nature?

Negative supercoiling twists DNA to make it more accessible; predominates in nature; positive supercoiling is tighter and found in some archaeal extremes.

What is the nucleoid in bacteria?

The electron-dense, compacted region of bacterial DNA in the cytoplasm, achieved by DNA-binding proteins and ~10-fold compaction.

Which proteins compact bacterial DNA into the nucleoid?

HU, IHF, and H-NS (DNA-binding proteins).

What is the typical range of GC content in bacterial genomes and how does environment influence it?

Approximately 30–70% GC; higher GC content is often seen in organisms from warmer environments due to stronger G-C bonds.

What are plasmids and how do they differ from chromosomes in bacteria?

Plasmids are independent, extra-chromosomal DNA encoding nonessential genes; chromosomes carry essential genes (e.g., rRNA, tRNA, central dogma genes).

Name some common plasmid types and their functions.

Conjugative plasmids (transfer DNA via pili); virulence plasmids (virulence factors); resistance plasmids (antibiotic resistance); degradative/col plasmids (pollutant degradation/toxin genes).

What is an operon and where are operons common?

A group of two or more co-regulated genes transcribed as a single mRNA; common in bacteria and archaea.

What is the approximate genome size and gene count of E. coli K-12?

About 4.6 megabases; around 4,300 genes.

What is the genome size and gene count of Mycoplasma?

Approximately 0.58 megabases (580 kb); about 475 protein-coding genes.

Which organism has one of the largest known bacterial genomes and what is its size?

Syringium cellulosum; about 13 megabases; circular chromosome with ~10,000–12,000 genes.

What happens at the ter termination sites and what protein is involved in termination directionality?

Ter sites mark termination; Tus protein binds ter sites to stop replication; permissive vs non-permissive orientation determines whether termination proceeds.

What enzyme decatenates interlinked replicated chromosomes in bacteria?

Topoisomerase IV (Topo IV).

What proteins recognize and initiate archaeal replication origins, and how is archaeal replication similar to eukaryotes?

ORC1 and Cdc6 recognize origins; MCM helicase; replication resembles eukaryotic initiation with multiple origins and PCNA-like clamps.

Which polymerases are typical in archaea and what is the role of PCNA?

Pol D or Pol B as main archaeal polymerases; PCNA acts as the sliding clamp.

What enzyme is used in PCR and from which organism does it originate?

Taq polymerase from Thermus aquaticus.

What are the three main steps of a PCR cycle and their order?

Denaturation, annealing, extension.

Why are DNA primers used in PCR instead of RNA primers?

RNA primers are unstable at high temperatures; DNA primers are more stable and provide a 3'OH for extension.

What cofactor ions are required for DNA polymerase activity in PCR?

Magnesium (Mg2+) and zinc (Zn2+) in the active site.