Bacterial DNA replication & 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?

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

Which helicase unwinds DNA at the origin in bacteria?

DnaB

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

DnaC

What is the primosome in bacterial DNA replication?

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

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

DNA polymerase III 

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

DNA polymerase I

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

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

Links the two core DNA polymerases III and coordinates simultaneous leading- and lagging-strand synthesis; 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?

Topoisomerases

What is negative supercoiling?

Negative supercoiling twists DNA to make it more accessible; predominates in nature

What is the nucleoid in bacteria?

The 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

What is an operon?

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

What happens at the ter termination sites?

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

What proteins recognize and initiate archaeal replication origins?

ORC1 and Cdc6 recognize origins; MCM is the helicase

Which polymerases are typical in archaea?

Pol D or Pol B

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.

What protein initiates replication by binding to the OriC?

DnaA protein initiates replication by binding to the OriC

What is the function of Class I?

nick one strand to relieve torsion

What is the function of Class II Topoisomerase?

nick both strands and can generate/relax supercoils

What enzymes are Class I topoisomerase?

Topoisomerase I and III

What enzymes are Class II Topoisomerase?

Topoisomerase II and IV, including DNA gyrase

What is the function of PCNA?

It acts as the sliding clamp. It enhances the processivity of DNA polymerase during DNA replication.

what protein is involved in termination directionality?

Tus protein, which binds to ter sites.

Where are operons common?

common in bacteria and archaea

What would happen to replication if DnaA was knocked out?

OriC would not be recognized; DNA would not unwind at the origin; initiation of replication would not occur

What would happen if DnaC was disrupted?

helicase loading would be impaired, preventing DNA unwinding and replication initiation.

If DnaB helicase function was inhibited, what would happen?

DNA unwinding would be disrupted; parental DNA would not unwind; replication forks would stall; loss of replisome integrity

If dnaG primase was absent, what would happen?

no RNA primers would be synthesized, initiation of replication would be blocked, lagging strand synthesis would stop

If HU was deleted, what would happen?

oriC would not be properly opened, dnaA binding would fail, no helicase could load, replication initiation would be impaired

What would happen if the alpha subunit of DNA Pol III was inactivated?

no new nucleotides would be added; both leading and lagging strand synthesis would stop; proofreading could still occur

If the ε subunit of DNA Pol III was defective, what would happen?

all of the above

If the tau (t) subunit was missing, what would happen?

all of the above

If the B sliding clamp was disrupted, what would happen?

DNA pol III would frequently fall off DNA; replication would decrease, both leading and lagging strand synthesis would be impaired

If DNA Pol I was absent, what would happen?

RNA primers would remain in the DNA; lagging strand okazaki fragments could not be completed; nick translation would not occur

If DNA ligase was disrupted, what would happen?

Lagging strand Okazaki fragments would remain unsealed; nicks in the sugar-phosphate backbone would persist; DNA replication would be incomplete and lead to genomic instability.

If topoisomerase IV was inhibited, what would happen?

daughter chromosomes would remain catenated (linked) and chromosome segregation would fail during cell division

If the tus protein was absent, what would happen?

replication would not terminate properly; the replication forks could continue to progress

If DNA gyrase (topoisomerase II) was inhibited, what would happen?

positive supercoils ahead of the fork could not be relieved; helicase would stall; replication fork movement would stop; leading and lagging strand synthesis would stop

If RNase H activity was absent, what would happen?

RNA primers would remain on the lagging strand, preventing proper Okazaki fragment maturation and leading to incomplete lagging strand

What is positive supercoiling?

positive supercoiling is tighter and found in some archaeal extremes

What are bacteria packaging proteins?

HU, IHF, and HNS (DNA-binding proteins)

What is the role of topoisomerases in replication?

They prevent excessive supercoiling and relieve torsional strain during unwinding

Conjugative Plasmids

enable DNA transfer between cells via pili and play a crucial role in horizontal gene transfer

Virulence plasmids

encode factors involved in pathogenicity/virulence and contribute to a bacterium's ability to cause disease

Resistance plasmids

carry antibiotic resistance genes/proteins

Col plasmids

contain genes encoding bacteriocins/toxins targeting related species

Degradative plasmids

encode enzymes for pollutant degradation that enable bacteria to utilize harmful compounds as carbon or energy sources.

Plasmids are a major vehicle for horizontal gene transfer

True

Horizontal gene transfer

occurs between organisms of the same or different species

Vertical gene transfer

is the transfer of genetic material from a parent organism to its descendants, primarily occurring during reproduction

Replication is semi-conservative

True

Polymerization proceeds:

5’ to 3’

In bacteria replication is bidirectional from oriC and typically involves how many replication forks?

Two replication forks

What enzyme synthesizes RNA primers to provide 3’-OH for DNA polymerization?

dnaG (primase)

Primer loading is ATP-dependent

True

protect exposed ssDNA and prevent secondary structure formation

single-strand binding proteins (SSB proteins)

DNA polymerase always synthesizes the new DNA strand in the 5′ to 3′ direction

True

ParA/ParB

Involved in the segregation of plasmids during bacterial cell division, where ParA binds to DNA and ParB recognizes the centromere, ensuring even distribution of genetic material.

Primosome proteins

PriA, PriB, and PriC; they bind to the DNA

In the lagging strand synthesis, does the DNA pol III synthesizes the DNA in a 5’ to 3’ manner continuously or discontinuously?

Discontinuously

In the leading strand synthesis, does the DNA pol III synthesizes the DNA in a 5’ to 3’ manner continuously or discontinuously?

Continuously