slides 8

DNA Transfer in Bacteria

Overview of DNA Transfer Mechanisms

  • Conjugation: A process where bacteria exchange genetic material via direct contact.

  • Transduction: The transfer of bacterial DNA by a virus (bacteriophage).

  • Transformation: Uptake of free DNA from the environment by bacteria.

Homologous Recombination (HR) and DNA Repair

Formation of Recombinants by Homologous Recombination

  • Bacterial Genetic Structure

    • Bacteria are haploid organisms, which means they cannot maintain two copies of their chromosome.

    • Homologous recombination (HR) enzymes (such as RecA) assist in gene transfer and recombination events.

    • Conjugation often results in breakage of the mating bridge through which DNA is transferred.

    • Example approximation: The complete transfer of chromosomal DNA (~1 mm long) occurs over roughly 100 minutes.

General Roles of Homologous Recombination

  • Main Functions in Bacteria:

    • Essential for DNA repair and replication fork rescue.

    • Bacteria lack telomeres and meiosis, making HR critical for genomic stability.

Comparison: Mitosis vs. Meiosis and HR

  • Mitosis: Division of a parent cell into two identical daughter cells (2n).

  • Meiosis: Produces four haploid (n) daughter cells from one diploid parent (2n). Includes:

    • Prophase I: Duplicated chromosomes form tetrads through homologous recombination (crossing over).

    • Metaphase I: Chromosomes align at the metaphase plate.

    • Anaphase I: Homologous chromosomes are separated.

    • Anaphase II & Telophase II: Sister chromatids are separated in the second round of meiosis.

  • In contrast, bacteria recombine genetic material independently of meiosis.

Mechanism of Homologous Recombination

Requirements for HR

  • Two DNA segments must bear identical or highly similar sequences (either inter or intra genomic).

  • Synapsis occurs between the recombining regions.

  • Heteroduplex formation involves base pairing between the two segments, requiring four strands.

  • Resolution of the heteroduplex is achieved by cleaving and rejoining the DNA strands.

Models of Homologous Recombination

  • General Requirements:

    • Requires homologous sequences.

    • A crossover event is essential for genetic recombination.

  • Holliday Model (1964):

    • Describes the formation of two corresponding single-stranded breaks in two DNA molecules, followed by strand invasion and heteroduplex formation.

    • The subsequent isomerization leads to the formation of the Holliday junction.

    • Resolution can result in either crossover or non-crossover events.

  • Branch Migration: Facilitated by proteins and energy, branching effects base pairing extent in the heteroduplex.

Genetic Components Involved in Homologous Recombination (E. coli)

  • Table 9.1 Major Genes Encoding Recombinational Functions

    • recA: Critical for synapsis and strand invasion.

    • recBC, recD: Help initiate recombination by unwinding DNA.

    • recF: Stimulates recA loading at single-strand gaps.

    • Holliday Junction Resolving Enzymes (ruvA, ruvB, ruvC): Control migration and resolution of Holliday junctions.

Reverse Genetics Applications

  • Homologous recombination makes reverse genetics in bacteria and lower eukaryotes effective by allowing specific genetic modifications.

Gene Function and Mutant Analysis

Isolation and Identification of Rec- Mutants

  • Assumption: Mutations in recombination-related genes prevent HR.

    • A Rec- mutant will not produce Arg+ Trp+ transconjugants when using Hfr strains.

    • Mutagenesis of an Arg+ Trp- strain (F-) can help identify Rec- mutants based on their inability to transfer specific genes.

Summary of Key Points in HR Mechanics

  • Bacteria utilize homologous recombination effectively without meiosis.

  • Models of homologous recombination (Holliday model vs. alternative models) are crucial for understanding genetic exchanges.

  • Key proteins like RecA facilitate the processes involved in HR.

  • Mutant isolation, particularly Rec- mutants, helps elucidate the underlying mechanics of recombination processes.

DNA Damage and Repair Mechanisms

Causes of DNA Damage

  • DNA lesions occur independently of DNA replication, caused by various factors such as:

    • Missing bases

    • Altered bases from ionizing radiation

    • Strand breaks due to chemical interactions.

DNA Repair Pathways in E. coli

  • Different repair mechanisms address specific types of DNA damage.

  • Enzymes involved include:

    • Methyl-directed mismatch repair (mut proteins).

    • Nucleotide excision repair (uvr proteins).

    • Base excision repair (xthA, nfo).

    • Single-stranded gaps repaired by recombination processes (recA).

The SOS Response

  • The SOS response serves as a mechanism for bacterial survival under extreme DNA damage.

    • Induced by severe damage, leading to expression of error-prone repair genes.

    • Operates under LexA regulation, where LexA represses SOS genes under normal conditions but is cleaved by RecA upon DNA damage.

Mutagenic Repair Genes

  • Genes umuC and umuD are implicated in error-prone repair pathways, facilitating translesion synthesis (TLS) under damage conditions.

  • Understanding these pathways and the effective mechanism of mutagenic repair is critical for assessing genetic stability in prokaryotes and advancements in genetic engineering applications.