DNA Repair: Clamping down on mismatches
- University of Southern California Keck School of Medicine, United States
Errors made when DNA is replicated can lead to cancer and genetic disorders, so cells rely on a system called mismatch repair (MMR) to remove many of the errors from newly synthesized DNA (Hombauer et al., 2011; Kunkel and Erie, 2015; Simmons et al., 2008). These errors include mismatches that occur when the wrong DNA base is added into the new DNA strand (i.e. a base that does not match the one in the template strand). MMR must deal with these mistakes before the DNA is repackaged into nucleosomes, which would block the repair mechanisms. As such, a long-standing question in this field is how the MMR system discriminates the new strand (which contains the errors) from the template (which is error-free) both quickly and accurately.
Now, in eLife, Tatsuro Takahashi and colleagues at Osaka University and Kyushu University report a new twist in this story (Kawasoe et al., 2016). Newly synthesized strands of DNA have small gaps or nicks (Kunkel and Erie, 2015), and such signals can be used to direct MMR to a specific DNA strand in cell-free experiments (Holmes et al., 1990). However, Takahashi and colleagues – who include Yoshitaka Kawasoe as first author – found, via experiments with Xenopus cell extracts, that MMR can still correctly identify the new DNA strand even after any gaps or nicks had been filled in. This unexpectedly implied that there must be a second signal that allows MMR in eukaryotes to distinguish the new DNA strand from the template.
So, what is this second signal? PCNA is a ring-shaped complex that acts as a “sliding clamp” to coordinate DNA replication by traveling along the DNA template. Eukaryotes use PCNA and a mismatch recognition complex called MutSα to start MMR (Kadyrov et al., 2006; Kunkel and Erie, 2015; Umar et al., 1996), and these two complexes interact directly at the site of a mismatch (Clark et al., 1999; Flores-Rozas et al., 2000; Kleczkowska et al., 2001). Kawasoe et al. now reveal that the PCNA complex provides the secondary signal that allows the cell to “remember” which strand of DNA is which (Figure 1A,B).
Figure 1
PCNA binds to DNA in an asymmetric way.
Base-base mismatches (dashed circle) result when a base is added into a newly synthesized DNA strand (red) that does not match the corresponding base in the template strand (blue). (A) Newly synthesized strands of DNA often have small gaps that are filled in and ligated later, and the ring-shaped complex PCNA forms non-identical complexes with 5’-gapped (top) or 3’-gapped (bottom) heteroduplex DNA. Note that one side of the complex (green) always faces towards the 5’ end of the new DNA strand, while the other (yellow) faces towards the 3’-end. (B) The asymmetry of the PCNA-DNA complexes is conserved after the DNA gaps are filled in and ligated; this could allow DNA mismatch repair to distinguish the error-containing new strand from the error-free template. (C) Kawasoe et al. show that PCNA spends little time on DNA containing a mismatch in the absence of the mismatch recognition complex MutSα (top). PCNA spends much longer on the DNA when MutSα is present (middle), but not quite so long if MutSα’s PCNA interacting protein (PIP) motif is removed (MutSα∆PIP; bottom). Times given in figure are the half-lives of the PCNA-DNA complexes (t1/2).
The PCNA clamp loads onto DNA ends in an asymmetric way (Pluciennik et al., 2010). This means that one side of the ring complex always faces towards the 5’-end of the newly synthesized DNA, while the other faces towards its 3’-end. Kawasoe et al. propose that this inbuilt asymmetry is used to direct the enzymes that correct mismatches towards the new strand and not the template strand.
Kawasoe et al. also found that MutSα strongly encourages the PCNA clamp to remain loaded on the DNA. They showed that this effect was much weaker if MutSα lacked the domain that it uses to interact with PCNA (Figure 1C). These findings further suggest that the interaction between MutSα and PCNA might act in favor of MMR, rather than DNA replication. If, as Kawasoe et al. suggest, the asymmetric PCNA-DNA complex forms a biological “memory” of which DNA strand is which, then the interaction between MutSα and PCNA appears to make that memory more stable over time, similar to converting short-term memories to long-term ones.
However, several questions remain to be answered. For example, the PCNA asymmetry and its interaction with MutSα may create a long-term “memory” for strand-specific MMR, but why do cells delay the packing of DNA into nucleosomes? Also, do the various modifications that are made to PCNA (such as ubiquitination and phosphorylation; Ortega et al., 2015) alter its role in strand-discrimination in MMR? Finally, does MMR in bacteria also use β clamp (the bacterial counterpart of PCNA) in the same way (Simmons et al., 2008)? Additional studies are now needed to answer these important questions.
References
-
Functional analysis of human MutSalpha and MutSbeta complexes in yeastNucleic Acids Research 27:736–742.https://doi.org/10.1093/nar/27.3.736
-
Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell linesProceedings of the National Academy of Sciences 87:5837–5841.https://doi.org/10.1073/pnas.87.15.5837
-
hMSH3 and hMSH6 interact with PCNA and colocalize with it to replication fociGenes & Development 15:724–736.https://doi.org/10.1101/gad.191201
-
Eukaryotic Mismatch Repair in Relation to DNA ReplicationAnnual Review of Genetics 49:291–313.https://doi.org/10.1146/annurev-genet-112414-054722
-
Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesisProceedings of the National Academy of Sciences of the United States of America 112:5667–5672.https://doi.org/10.1073/pnas.1417711112
-
PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repairProceedings of the National Academy of Sciences of the United States of America 107:16066–16071.https://doi.org/10.1073/pnas.1010662107
Article and author information
Author details
Publication history
Copyright
© 2016, Li
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,168
- views
-
- 148
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
DNA Repair: Clamping down on mismatches
eLife 5:e18365.
https://doi.org/10.7554/eLife.18365
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 Å-resolution X-ray structure that revealed a highly α-helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.
-
- Biochemistry and Chemical Biology
- Cell Biology
Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.
