Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry	Spring 2002
Lecture Notes:: 6 March	© R. Paselk 2002
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DNA Replication

DNA replication is:

• Semiconservative (each replicated double helix consists of one parental strand and one new strand).
• Bidirectional
  • DNA has two anti-parallel strands (one 5'-3' the other 3'-5')
  • Replication of each strand occurs in the 5'-3' direction.
• The double-helix must be unwound in order to expose bases for base-pairing and allow polymerizing enzymes access.
• Semi-discontinuous
  • Both strands are synthesized at each replication fork.
  • DNA polymerases synthesize DNA only in the 5'-3' direction, while reading the template in the 3'-5' direction.
  • One strand, the leading strand, is continuously replicated 5'Æ3' (reading 3'Æ5').
  • The opposite, lagging strand, is copied discontinuously as sufficient length becomes exposed to also synthesize in a 5'-3' direction.
    • The resulting, short ssDNA pieces (1000-2000 nucleotides) are called Okazaki fragments.
    • These fragments are then joined to form a continuous strand.

DNA Polymerases

All known DNA polymerases, both from prokaryotes and eukaryotes, share a common characteristics:

• The complementary bases are selected within the polymerase active site.
• DNA is synthesized in the 5'Æ3' direction, antiparallel to the template strand.
• A primer is required. That is the new DNA strands must be added to an existing free 3'-OH group (can be on DNA or RNA).
• All four d NTPs and Mg²⁺ are required in the reaction media.

E. coli Polymerases

There are three DNA polymerases in E. coli. Some of the properties of these enzymes are summarized in the table:

E. coli DNA Polymerase Properties Pol I Pol II Pol III (core) Mass (kD) 103 90 130 (a), 27.5 (e), 8.6 (q) Molecules/cell 400 ? 10-20 Turnover number* 600 30 1200-9000 Processivity 20 ? 10-15 (holoenzyme >5,000) Gene polA polB polC, dnaQ, holE Polymerization 5'Æ3' 5'Æ3' 5'Æ3' Exonuclease 3'Æ5' & 5'Æ3' 3'Æ5' 3'Æ5' * nucleotides polymerized/min/active site @ 37° C

DNA pol I: This is the first polymerase discovered and characterized. It serves as a model for the others because it is well understood. Pol I has three active sites on a single peptide chain. The protein, with a single 928 residue peptide chain, can be cleaved into two parts, a 323 residue smaller fragment which carries the 5'-3' exonuclease activity and a larger 605 residue piece known as the Klenow fragment which carries the polymerase and 3'-5' exonuclease activity in a single cleft with two widely separated active sites.

As seen in the table Polymerase I has three activities:

• 5'Æ3' Polymerase catalyzing the nucleophilic attack of the 3'-OH of the growing chain on the a-P of an NTP. It requires:
  • All four dNTPs
  • A primer with a free 3'-OH group
  • A template
  • Mg²⁺
• The 3'-5' exonuclease enables the enzyme to proofread the growing DNA chain as it is polymerized. It turns out that the polymerase cannot readily elongate an improperly base-paired terminus on the growing chain. Thus when an improper base-pair is formed polymerization ceases until the exonuclease site has a chance to remove it, allowing continued growth. This proofreading activity accounts for part of the high fidelity of DNA replication as compared to other polymerization enzymes.
• The 5'-3' exonuclease activity on the other hand enables pol I to edit DNA double strands by nick translation. In this situation the pol I enzyme can bind to a nick (a single strand break) and remove bases in the 5'-3- direction, while laying down a new set of bases using its 5'-3' polymerization activity. This is particularly useful for removing RNA base-paired to DNA and for removing segments of DNA involving errors (such as thymine dimers formed by exposure to UV light).

Note that pol I cannot be the main polymerization enzyme because it is way too slow, and shows a low processivity (it falls off the DNA easily making polymerization of large DNA molecules difficult).

DNA pol III functions as a holoenzyme in vivo. It both labile and complex. The subunit composition for the E. coli enzyme is shown in the table below.

E. coli DNA Polymerase III Subunits Subunit Mass (kD) Gene Function a 130 polC polymerase e 27.5 dnaQ 3'-exonuclease q 8.6 holE a, e assembly t 71 dnaX holoenzyme assembly on DNA b 41 dnaN Sliding clamp-greatly increases processivity. g 47.5 dnaX(Z) g-complex d 39 holA g-complex d' 37 holB  g-complex c 17 holC  g-complex y 15 holD  g-complex

Pol III can also be isolated as a core enzyme. In this form it will catalyze polymerization, however its processivity drops from the >5000 bps in the holoenzyme to about 10-15 bps. The holoenzyme also requires ATP to bind to the template/primer.

Pol III differs from Pol I in that it cannot unwind DNA, requiring a series of initiation proteins, unwinding proteins, single stranded binding protein (ssb), etc. as shown in the table below. These work in concert with ATP hydrolysis (2 ATP/bp) to open the DNA double helix in advance of Pol III. Note that the ssb must be stripped off of the DNA strands before Pol III can replicate it.

The actual replication of DNA occurs in the replisome, a complex including two Pol III enzymes, one to synthesize the leading strand and one to synthesize the lagging strand. This results in a difficulty since the leading strand is replicated only in the 5'-3' direction, and the complimentary strand is then going the opposite direction. This problem is solved by synthesising a sufficient length of DNA to allow the lagging single strand to loop around and come in parallel, instead of anti-parallel, to the leading strand. Note that after each Okazaki fragment is synthesized by the lagging Pol III, the enzyme must relocate along the lagging strand, by opening and closing the lagging "clamp" to a newly synthesized primer to start the next fragment.

E. coli DNA Replication Proteins Protein Function DNA polymerase III holoenzyme DNA synthesis  Replication Initiation Proteins DNA gyrase DNA unwinding (relieves supercoiling induced by replication and DnaB) ssb single-stranded binding protein (prevent ssDNA from reannealing behind helicase) DnaA Initiation factor (Multimeric complex binds at oriC and causes helix to open [melt] with ATP hydrolysis.) HU DNA binding (histone-like). Prevents non-oriC binding of DnaA Primosome (required to initiate each Okazaki fragment) PriA Primosome assembly, 3'Æ5' helicase PriB Primosome assembly PriC Primosome assembly DnaB 5'Æ3' helicase, unwinds DNA in ATP dependent manner, producing positive supercoiling. Part of prepriming complex. DnaC Delivers DnaB to oriC DnaT Assists DnaC in delivery of DnaB Primase (DnaG) Synthesizes RNA primer DNA pol I Removes RNA primer, replacing with DNA. Tus Termination of polymerization at Ter locus opposite oriC

Synthesis of the lagging strand also requires two additional enzymes:

• DNA Pol I to hydrolyze off and replace the RNA primers formed by the Primosome.
• Ligase to seal the remaining gaps in this strand. The ligase reaction does require a source of free energy, which is supplied differently in the prokaryote and eukaryotes studied:
  • E. coli uses NAD⁺ hydrolysis as the source of energy. Note that the reaction involves the phosphoric acid anhydride bond in NAD⁺.
    • The energy in this bond is initially captured in a phosphoamide bond with an active site lysine residue, with the release of NMN⁺
    • The "AMP" portion is then transferred from the lysine nitrogen to the 5'-P of the nick to form a new phosphoric acid anhydride bond, again capturing the energy.
    • Finally the 3'-OH group attacks the 5'P, displacing AMP and closing the nick.
  • Eukaryotes use ATP instead of NAD⁺ as their source of the "AMP" residue, releasing PP_i instead of NMN⁺. Otherwise the reactions are the same.

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Last modified 12 March 2002