Figure 8.2 Objectives: Identify the key molecular players involved in DNA replication Construct a sequence of events that summarizes the process of DNA replication Compare and contrast leading strand versus lagging strand synthesis DNA Replication

Polymer of nucleotides: adenine, thymine, cytosine, guanine Double-stranded/Double helix "Backbone" of each strand is deoxyribose-phosphate Strands held together by hydrogen bonds between AT and CG Strands are antiparallel DNA: A Review Figure 8.4 What do we already know about DNA?

LE 16-9_4 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. The nucleotides are connected to form the sugar-phosphate back- bones of the new strands. Each “ daughter ” DNA molecule consists of one parental strand and one new strand. DNA Replication Is Semiconservative

Origin of replication 5’ 3’ 5’ Frame of reference

Origin of replication 5’ 3’ 5’ Frame of reference DnaA proteins ( ) – initiate replication

Origin of replication DnaA proteins ( ) – initiate replication 5’ 3’ 5’ Replication bubble – binding generates a “bubble” allowing for bidirection replication

Helicase enzyme ( ) – unwinds the double helix 5’ 3’ 5’ – replication bubble expands Replication forks (expanding in opposite directions)

Helicase enzyme ( ) – unwinds the double helix 5’ 3’ 5’ – replication bubble expands Single-strand binding proteins ( ) – keep the strands separated

Helicase enzyme ( ) – unwinds the double helix 5’ 3’ 5’ – replication bubble expands Single-strand binding proteins ( ) – keep the strands separated Gyrase ( ) – relieves supercoiling ahead of replication fork

Primase enzyme ( ) – builds a short RNA segment 5’ 3’ 5’

Primase enzyme ( ) – builds a short RNA segment 5’ 3’ 5’ – RNA serves as a “primer” ( )

5’ 3’ 5’

Primase enzyme ( ) – builds a short RNA segment 5’ 3’ 5’

3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand Primase enzyme ( ) – builds a short RNA segment – RNA serves as a “primer” ( )

DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group 5’ 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand

5’ 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

5’ 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

5’ 3’ 5’ = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

LE New strand 5 end Phosphate Base Sugar Template strand 3 end 5 end 3 end 5 end 3 end 5 end 3 end Nucleoside triphosphate DNA polymerase Pyrophosphate

= RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’

= RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ Primase enzyme ( ) – builds a short RNA segment

= RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ Primase enzyme ( ) – builds a short RNA segment – RNA serves as a “primer” ( ) When DNA Polymerase III encounters a road block, it disengages and starts building from a newly generated primer

= RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

= RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ – builds complementary strand in 5’  3’ direction DNA Polymerase III ( ) – extends from the RNA primer’s available 3’ hydroxyl group

= RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Leading strand synthesis always heads toward its respective replication fork Lagging strand synthesis always heads away from its respective replication fork

Process of leading strand synthesis is called “continuous” Process of lagging strand synthesis is called “discontinuous” = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Leading strand synthesis always heads toward its respective replication fork Lagging strand synthesis always heads away from its respective replication fork

Disconnected segments resulting from discontinuous synthesis are called Okazaki fragments = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Okazaki framents

RNA primers are replaced with DNA nucleotides using DNA polymerase I = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’

RNA primers are replaced with DNA nucleotides using DNA polymerase I = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ DNA Polymerase I ( ) – uses neighboring fragment’s free 3’ hydroxyl to extend and displace RNA nucleotides

RNA primers are replaced with DNA nucleotides using DNA polymerase I = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ DNA Polymerase I ( ) – uses neighboring fragment’s free 3’ hydroxyl to extend and displace RNA nucleotides 5’

RNA primers are replaced with DNA nucleotides using DNA polymerase I = RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ DNA Polymerase I ( ) – uses neighboring fragment’s free 3’ hydroxyl to extend and displace RNA nucleotides 5’

= RNA primer for leading strand = RNA primer for lagging strand 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ DNA Ligase ( ) – joins disconnected neighboring fragments

= RNA primer for leading strand = RNA primer for lagging strand 3’ 5’ 3’ 5’ DNA Ligase ( ) – joins disconnected neighboring fragments – yields one contiguous piece of newly synthesized complementary DNA