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My copy of the Molecular Biology of the Cell (the 5th edition, so not entirely up to date) seems to claim that exonuclease action is an important part of DNA proofreading, and that dysfunction in this leads to an increased rate of mutation (pp 276-277).

But at the same time it specifies that exonuclease action is only useful in removing incorrect bases from DNA-primers (p. 270). That means that only the base pairs in the one primer on the leading strand would be proofread, and only a minority of the base pairs in the lagging strand (The primers are vastly more numerous, but still only a minority of base pairs will be found inside primers).

In addition, these primers are made out of RNA and are only temporary, so I can’t see why mutations in these sequences would be considered important. They are replaced with DNA-segments at the end anyway and it’s these final DNA-segments that ought to be proofread.

So how can exonuclease action be considered an important (or even vaguely relevant) proofreading mechanism?

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  • $\begingroup$ If you could find and provide links to the corresponding sections in the fourth edition available to all at NCBI Bookshelf on line, we could check whether you are misinterpreting the text. Few of us will have a physical copy to relate to your page numbers. Otherwise decent sized quotations might help. $\endgroup$
    – David
    Oct 13 at 7:39
  • $\begingroup$ @David Updated text with exerpts, this was somewhat labour intensive ^^ $\endgroup$
    – Magnus
    Oct 13 at 8:53
  • $\begingroup$ @David .....and those changes just disappeared somehow (different versions open in different tabs?) $\endgroup$
    – Magnus
    Oct 13 at 9:22
  • $\begingroup$ You have my sympathies. Sometimes I pause in the middle of an answer or edit and... I'll have a look myself when I have a moment. $\endgroup$
    – David
    Oct 13 at 11:34
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    $\begingroup$ I have edited your question to change "exonucleases" to "exonuclease action". DNA polymerase has both polymerization and exonuclease action, and it is this that the section you mention is referring to. This is evident in the diagram in my answer, which is therefore confined to your main problem — confusion about the usage of the term "primer". $\endgroup$
    – David
    Oct 15 at 7:34
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Without access to the 5th edition of The Molecular Biology of the Cell I am relying on the section in the 4th edition which describes proofreading, and specifically Figure 5.9, which I have reworked in a linear manner so as not to occupy too much space on the page:

DNA exonucleic proofreading

If this is, indeed, the section to which the poster refers, the problem is one of misunderstanding the term primer.

The poster appears to be thinking of the priming of DNA synthesis at the replication fork by RNA — Okazaki pieces to initiate copying of the template strand. However this specific usage of primer is not that which is meant in the diagram shown. Long before Okazaki discovered RNA priming of DNA synthesis, analysis of the reaction of DNA polymerase in vitro had shown that the process required 1. A template and 2. A primer with a 3′-OH end, with which the 5′-dNTP phosphate could react. And I for one (for I am very old) taught that to students.

So what the authors are referring to is the continual extension of the DNA strand, not its initiation. And the primer is the 3′-OH end of the growing complementary strand, hydrogen bonded to the template strand.

Appendix: Historical Perspective of the usage of ‘primer’

The double-stranded structure of DNA in 1953 suggested a ‘inverse copying’ mechanism to maintain the genetic information of an organism on cell division. A DNA template was obviously assumed, but it was not anticipated that the enzymes responsible for this (DNA polymerases) would have any further requirements. It was therefore unanticipated that the DNA polymerase described by Arthur Kornberg in 1959 could only ‘copy’ a DNA template if there was already a base-paired portion of DNA on the a complementary strand with a 3′-OH end with which the dNTPs reacted. The same was true of every other DNA polymerase subsequently isolated, and contrasted with the DNA-dependant RNA polymerases, which did not have this restriction. This pre-existing DNA on the complementary strand was termed a ‘primer’, with no prejudice of length implied. For the next twenty years students were taught that the three salient features of DNA polymerase were that it required a template, a primer with a 3′-OH end, and used dNTPs. And nobody could explain how initiation occured, and more saliently, how synthesis on one of the strands occured in the 3′ to 5′ direction.

It was only in the early 1980s that the repeated generation of small stretches of complementary RNA at the replication fork provided the 3′-OH ends that DNA polymerase could extend from. The term ‘primer’ was naturally applied to these complementary RNA stretches, but (especially as the RNA was later removed) with time became associated with the fragments (Okazaki fragments) being of small size. This association was further reinforced with the advent of the polymerase chain reaction which uses small synthetic oligonucleotide primers, as do other molecular genetic techniques.

The first edition of The Molecular Biology of the Cell was published in 1983, and its authors had worked in the scientific environment of the 60s and 70s, and naturally used the terminology of that era. Perhaps writing today, they might have used a term such as “elongating strand”, rather than “primer strand”, to avoid confusing a different generation of young scientists.

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  • $\begingroup$ Thank you for your time and effort! $\endgroup$
    – Magnus
    Oct 16 at 6:38
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    $\begingroup$ Not at all. I have added a historical appendix, which puts your confusion in perspective (and makes me feel very old). $\endgroup$
    – David
    Oct 17 at 17:03

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